Novel compounds with high therapeutic index

ABSTRACT

The present invention is directed to novel therapeutic compounds comprised of an amino acid bonded to a medicament or drug having a hydroxy, amino, carboxy or acylating derivative thereon. These high therapeutic index derivatives have the same utility as the drug from which they are made, and they have enhanced pharmacological and pharmaceutical properties. In fact, the novel drug derivatives of the present invention enhance at least one therapeutic quality, as defined herein. The present invention is also directed to pharmaceutical compositions containing same.

RELATED APPLICATION

This is a continuation of copending application U.S. Ser. No.11/343,557, filed on Jan. 30, 2006, which in turn is acontinuation-in-part of PCT Application No. PCT/US04/24901 which claimsbenefit of provisional application No. 60/491,331 filed on Jul. 29,2003.

SCOPE OF THE INVENTION

The invention relates to amino acid derivatives of pharmaceuticalcompounds, methods of treating particular ailments, which areameliorated by the administration of these drugs and pharmaceuticalcompositions containing these drugs.

The current invention involves improving many physicochemical,biopharmaceutical, and clinical efficacy of various drugs using aminoacids as covalently bonded carriers for these drugs.

BACKGROUND OF THE INVENTION

The development of chemical compounds for the treatment of disorders,maladies and diseases has become increasingly difficult and costly. Theprobability of success for such development is often discouragingly low.Further, the time for development can approach or exceed ten years,leaving large numbers of patients without remedy for an extended periodof time.

Even in cases in which effective pharmaceutical compounds have beendeveloped, there are often disadvantages associated with theiradministration. These disadvantages can include aesthetic, andpharmacokinetic barriers affecting the effectiveness of some existingpharmaceutical compounds, For example, unpleasant taste or smell of apharmaceutical compound or composition can be a significant barrier topatient compliance with respect to the administration regimen. Theundesirable solubility characteristics of a pharmaceutical compound canlead to difficulty in formulation of a homogeneous composition. Otherdisadvantages associated with known pharmaceutical compounds include:poor absorption of orally administered formulations; poorbioavailability of the pharmaceutical compounds in oral and othernon-intravenous formulations; lack of dose proportionality; lowstability of pharmaceutical compounds in vitro and in vivo; poorpenetration of the blood/brain barrier; excessive first-pass metabolismof pharmaceutical compounds as they pass through the liver; excessiveenterohepatic recirculation; low absorption rates from various sites,including skin, rectal, oral and buccal administration; ineffectivecompound release at the site of action; excessive irritation, forexample, gastro-intestinal irritability and/or ulceration; painfulinjection of parenterally administered pharmaceutical compounds andcompositions; excessively high dosages required for some pharmaceuticalcompounds and compositions, and other undesirable characteristics. Somepharmaceutical compounds are processed by the body to produce toxicby-products with harmful effects.

The art is continually seeking new chemical compounds for the treatmentof a wide variety of disorders, with improved properties to overcome thedisadvantages of known pharmaceutical compounds mentioned above.

The present invention has overcome many problems associated withcurrently marketed drugs by making amino acid derivatives. The conceptof such derivative chemistry is well known, and there are a number ofexamples of such derivatives enumerated in the literature and there andeven a few such derivatives are available in the market, including suchdiverse groups as statin drugs, ACE inhibitors, antiviral drugs such asAcyclovir and the like.

SUMMARY OF THE PRESENT INVENTION

The present invention, however, uses amino acids as the moiety to makesuch derivatives. The inventor has found that the novel drug derivativesof the present invention have a number of advantages. For example, whenthe amino acid derivatives of the present invention are administered bya number of routes such as oral, IV, rectal or other such methods, theinventor has found that generally these derivatives are not broken downin vivo; rather the amino acid derivatives themselves possess thetherapeutic activity.

In many cases, the inventor found that the current amino acidderivatives do not dissociate into the drug from which the amino acidderivative is derived, since there appears to be no active drug in thesystemic circulation. Instead, what was found in the systemiccirculation was the amino acid derivative is active and less toxic thanthe corresponding drug and possesses high therapeutic index. Asignificant advantage of the amino acid derivative of the presentinvention is that it is non-toxic, and hence either assimilated into thebody or safely excreted. This is quite unlike the characteristicsexhibited by a number of other drug derivatives available in the market,where the promoiety itself is toxic, as is the case with statin drugs,Enalapril, Benazapril and the like group of ACE inhibitors, and a numberof antibiotics such as pivoxil, Axetil, Cilexetil and the like groups,which are highly toxic, thereby reducing the therapeutic index of theactive drug.

On the other hand, the amino acid derivatives of the present inventionalso impart a number of advantages as shown herein below.

The present invention is directed to pharmaceutically active drugs,having an amino acid covalently bonded thereto to form said amino acidderivative, which is administered in this form to the subject, such as amammal.

The amino acid is an ideal model to be used as a derivative, because itis capable of forming various types of linkages between itself and thedrug. By definition, an amino acid has at least two functionalitiesthereon, an amino group (NH₂) and a carboxy group (COOH). For example,the α-amino acids have the well known structure

wherever R₀ is the side group or chain of the amino acid. The

as defined herein, is the main chain of the amino acid. Thus, forexample, in addition to the amino group and the carboxyl group on themain chain, the side chain may have other functional groups thereon suchas SH or OH or NH₂ and the like. The functional groups on the amino acidmoiety permit the covalent linkage to occur between the amino acid andthe drug.

As defined herein, the drug or medicament useful in the presentinvention contains functional groups thereon that permit the drug toreact with and form a covalent bond with the amino acid. Examples offunctional groups present on the drugs include NH₂, OH, COOH oracylating derivatives (acid derivatives) thereof, such as esters,amides, anhydrides and the like.

The mode of attachment between the pharmaceutical compound and the aminoacid can be via:

1) An ester bond (—CO—O—) arising from condensation of a carboxylic acidand an alcohol or phenolic hydroxyl group, or throughtransesterification, for example:

-   -   a) Where the pharmaceutical compound has an aliphatic or        aromatic hydroxyl group, an ester bond can be formed with the        backbone carboxylic acid group of the amino group under        esterification conditions; or    -   b) Where the pharmaceutical compound has an aliphatic or        aromatic hydroxyl group and the amino acid has a side chain        carboxylic acid group, an ester bond can be formed therebetween        under esterification conditions; or    -   c) Where the pharmaceutical compound has a carboxylic acid group        and the amino acid has a side chain aliphatic or aromatic        hydroxyl group, an ester bond can be formed therebetween under        esterification condition; or    -   d) Where the pharmaceutical compound has an ester group with        substituted or unsubstituted acyloxy (e.g., carbonylalkoxy or        arylalkoxycarbonyl, or aryloxycarbonyl) substituent        (compound-O—CO-substituent) and the amino acid has a backbone        carboxylic acid group, an ester bond can be formed therebetween        through transesterification; or    -   e) Where the pharmaceutical compound has an ester group with a        substituted or unsubstituted acyloxy (e.g., alkoxycarbonyl or        arylalkoxycarbonyl, or aryloxy carbonyl) substituent        (compound-O—CO-substituent) and the amino acid has a side chain        carboxylic acid group, an ester bond can be formed therebetween        through transesterification; or    -   f) Where the pharmaceutical compound has an ester group with a        substituted or unsubstituted alkoxycarbonyl or        arylalkoxycarbonyl or aryloxy carbonyl substituent        (compound-CO—O-Substituent) and the amino acid has a side chain        aliphatic or aromatic hydroxyl group, an ester bond can form        therebetween though transesterification; or    -   g) The alcohol and carboxylic acid moieties may be on the same        molecule such they can form an internal ester. For example,        certain compounds like Gabapentin can form an internal ester        under ester forming conditions, and is also included within the        scope of the present invention.

2) An amide bond (—CO—NH—) arising from condensation of a carboxylicacid and an amine, for example:

-   -   a) Where the pharmaceutical compound has an amino group and the        amino acid has a backbone carboxylic acid group, an amide can be        formed under amide forming conditions; or    -   b) Where the pharmaceutical compound has an amino group and the        amino acid has a side chain carboxylic acid group, an amide bond        can form therebetween under amide forming conditions; or    -   c) Where the pharmaceutical compound has a carboxylic acid group        and the amino acid has a backbone amino group, an amide bond can        form therebetween under amide forming conditions; or    -   d) Where the pharmaceutical compound has a carboxylic acid group        and the amino acid has a side chain amino group, an amide bond        can be formed therebetween under amide forming conditions.

Thus, the present invention is directed to the amino acid derivativesthus formed. As shown hereinbelow, the amino acid derivatives describedherein have advantages not realized relative to the drug without theamino acid attached thereto. For example, it can improvebioavailability, efficacy, be less toxic, exhibit greater solubility inwater and/or improve the ability of the drug to pass into the cellmembrane or through blood brain barrier, exhibit less side effects, suchas gastro-intestinal irritability, enhanced therapeutic index and thelike.

Thus, the present invention is directed to a method of improving thetherapeutic quality of a drug wherein the improvement in the therapeuticquality is selected from the group consisting of improved efficacy,enhanced therapeutic index, increased solubility in the mammal'sinternal fluid, improved passage through the cell membrane, improvedpassage through the blood brain barrier, decreased side effects, such assignificantly decreased irritation and/or ulcerations, less toxicity,enhanced absorption ratio and the like relative to the correspondingdrug administered to the patient in the derivatized form, said methodcomprising reacting the drug with an amino acid to form a covalent bondtherebetween and administering the product thereof (hereinafter“derivative”) to a patient. The amino acid derivatives of the presentinvention have at least one improved quality. In fact, they exhibitpreferably at least one of the improved qualities cited hereinabove, andmore preferably, at least two of the qualities described herein. Otheradvantages of the derivative include the wide availability of the aminoacids and the ease in which the reactions take place. The reaction toform the amide and esters are generally efficient and yields are veryhigh, presumably above about 60% and more preferably above about 75% andmost preferably above about 90%.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent application contains at least one drawingsexecuted in color. Copies of this patent application with colordrawing(s) will be provided by the United States Patent and TrademarkOffice upon request of the necessary fee.

FIG. 1 graphically compares the efficacy of L-serine ester of (±)Ibuprofen (F1), L-threonine ester (±) Ibuprofen (F2) andL-hydroxyproline ester of (±) Ibuprofen (F3), (±) Ibuprofen (i.e., theracemic mixture) and Ibuprofen (S)(+), after one hour dosing, based onthe antagonizing property of Acetylcholine induced writhes in Albinomice.

FIG. 2 graphically compares the efficacy of L-serine ester of (±)Ibuprofen, (F1), L-threonine ester of, (±) Ibuprofen (F2),L-hydroxyproline ester of (±) Ibuprofen (F3), ±Ibuprofen and S(±)Ibuprofen after 3 hour dosing, based on the antagonizing property ofAcetylcholine induced writhes in albino mice.

FIG. 3 depicts graphically the dose response relationship to meanclotting time (MCT) in minutes for the L-serine ester of acetylsalicylicacid (Formulation 1).

FIG. 4 depicts graphically the dose response relationship to meanclotting time (MCT) minutes for the L-hydroxyproline ester ofacetylsalicylic acid (Formulation 2).

FIG. 5 depicts the dose response relationship to mean clotting time(MCT) in minutes for the L-threonine ester of acetylsalicylic acid(Formulation 3)

FIG. 6 depicts the dose response relationship to mean clotting time(MCT) in minutes for control (acetylsalicylic acid).

FIG. 7 graphically compares the relative efficacy of L-serine (ester ofacetyl salicyclic acid (F.1), L-threonine ester of acetyl salicylic acid(F.2), L-hydroxyproline ester of acetylsalicylic acid (F.3), andacetylsalicylic acid (PC) as a function of mean clotting time inminutes.

FIG. 8 compares graphically the average clotting time in minutes ofAcetyl salicyclic Acid L-Threonine Ester and BAYER Aspirin at 325 mg and81 mg.

FIG. 9 compares in a bargraph the clotting time in minutes after 5 dayadministration of 81 mg Acetyl Salicyclic Acid L-Threonic Ester andBayer Aspirin in 3 human volunteers, in which two volunteers took theAcetyl Salicyclic Acid L-Threonine Ester and the third took one tookjust the Bayer Aspirin.

FIG. 10 compares graphically the percentage increase in clotting timeafter a 5 day administration to a patient of Acetylsalicyclic AcidL-Threonine Ester relative to Aspirin (Bayer) at 81 mg dose. The plotwas derived from the data in Table 29. Two patients tookAcetylsalicyclic Acid L-Threonine Ester b over a period of five days.The third volunteer took Bayer Aspirin for 5 days.

FIG. 11 plots the concentration of Aspirin versus time in fourvolunteers who took acetyl salicylic Acid L-Threonine Ester and twovolunteers who took Bayer Aspirin.

FIG. 12 is a plot of the plasma concentration of salicyclic acid inhumans plasma with respect to 4 of volunteers who were administeredAcetyl-salicuclic Acid L-Threonie Ester and two volunteer who wereadministered Aspirin.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

As used here, the terms “drug”, “medicament”, and “pharmaceutical” and“active agent” are being used interchangeably and refer to the activecompound that is administered to the patient without attachment of theamino acid thereto. Moreover, as used herein, the drug contains afunctional group thereon capable of reacting with the amino acid, suchas NH₂, OH, COOH or acylating derivatives thereof (e.g., ester,anhydride, amide, and the like) and the like. When the drug is linked toan amino acid, the term “amino acid derivative” or “derivative of thepresent invention” or synonym thereto is utilized.

A wide variety of therapeutically active agents can be used with thepresent invention. Examples include antacids; anti-inflammatorysubstances; coronary dilators; cerebaldilators; peripheral vasodilators;anti-invectives; psychotropics; anti-manics; stimulants;anti-histamines; laxatives; decongestants; vitamins; gastrointestinalsedatives; anti-diarreal preparations; anti-anginal drugs; vasodilators;anti-arrhythmics; anti-hypertensive drugs; analgesics; anti-pyretics;hypnotics; sedatives; anti-emetics; anti-nauseants; anti-convulsants;neuromuscular drugs; hyper- and hypoglycemic agents; thyroid andanti-thyroid preparations; diuretics, anti-spasmodics; uterinerelaxants; mineral and nutritional additives; anti-obesity drugs;anabolic drugs; erythropoietic drugs; anti-asthmatics; bronchodilators;expectorants; cough suppressants; mucolytics anti-uricemic drugs; drugsuseful for inhibiting organ transplant rejection; drugs useful fortreatment rheumatoid arthritis; antiviral agents; antibiotics; drugsuseful for treating asthma; drugs useful for treating urinary spasms orinfections; calcium channel blockers; drugs useful for treating malaria;drugs useful for treating skin diseases; anti-hyperlipidemics; drugsuseful for treating psychological diseases, such as, attention deficientdisorders, schizophrenia, and other psychoses, and the like; drugsuseful for treating tuberculosis; antidiabetics; anti cancer drugs;drugs useful for treating gastric hyperacidity; drugs useful fortreating eye diseases; and the like. However, the drug utilized must becapable of bonding to the amino acid. Consequently, it must have ahydroxy group, an amino group or a carboxy group or an acylatingderivative thereof. By acylating derivative it is meant that the drugcontains a carboxy group or a carboxylic acid derivative thereof, whichhas an acyl group thereon, and which can be hydrolyzed to the acid orsalt thereof, such as an amide, ester, anhydride, and the like.

The amino acids useful for reacting with the drugs are those containingthe free amino and/or carboxylic acid groups present in all conventionalamino acids. The preferred amino acids are described in more detailhereinbelow. They are preferably α-amino acids. In addition, they arepreferably L-amino acids and more preferably L-α-amino acids asdescribed hereinbelow. Of those, some preferred embodiments includethose amino acids having relatively high solubility in aqueous media,for example, in deionized water at unbuffered aqueous solution at 25°C., of at least 100 g/L, and more preferably, at least 250 g/L, and evenmore preferably at least 500 g/L. For example, glycine and proline havesolubilities in deionized water at 25° C. of approximately 250 g/L and1620 g/L, respectively, other preferred amino acids contain basic aminoside chains, such as lysine. For example, lysine has solubility indeionized water at 25° C. of approximately 700 g/L. Other preferredamino acids are those containing hydroxyl side chains, such ashydroxyproline, serine, and threonine. For example, threonine,hydroxyproline and serine have solubilities in deionized water at 25° C.of approximately 100 g/L, 369 g/L and 420 g/L, respectively. Otherpreferred embodiments include those amino acids with relatively lowsolubility in aqueous media, for example, in deionized water at 25° C.of at most 10 g/L, or for example, at most 2 g/L, or for example at most0.6 g/L. For example, the solubility of tyrosine in deionized water at25° C. is approximately 0.5 g/L. Such derivatives could be used toproduce formulations with extended release characteristics, due to thelimited solubility of the derivatives.

Additional preferred amino acids are those containing carboxylic acidside chains, such as glutamic acid and aspartic acid. However,non-essential amino acids, and the non-naturally occurring amino acidscan also be bonded to the drug, in accordance with the presentinvention.

The following reaction schemes depict the reactions discussedhereinabove with respect to the of hydroxyl, carboxyl and aminecontaining drugs with various amino acids. In the schemes below, R isthe drug less the functional OH, COOH or NH₂ group whichever is present,and R₁ is

wherein R₀ is the side chain of the amino acid listed hereinbelow:Reaction Scheme A: Where the hydroxyl group of the drug is reacted withthe carboxyl group of an amino acid to form the ester derivative

Reaction Scheme B: Where the carboxyl group of the drug is reacted withthe hydroxyl group of a hydroxy amino acid wherein the hydroxy group ison the side chain to form the ester derivative.

Reaction Scheme C: Where the amino group of the drug is reacted with thecarboxyl group of the amino acid to from the amide derivative

Reaction Scheme D: Where the carboxyl group of the drug is reacted withthe carboxyl group of the amino acid to form the anhydride derivative.

Reaction Scheme E: Where the amino group of the drug is reacted with theamino group of the amino acid to form the azo derivative derivative.

Reaction Scheme F: Where the carboxyl group of the drug is reacted withamino group of the amino acid to form the amide derivative.

In the above schemes A-F, the preferred amino acids used are shownhereinbelow:

As used herein the term “amino acid” refers to an organic compoundhaving therein a carboxyl group (COOH) and an amino group (NH₂) or saltsthereof. In solution, at neutral pH, these two terminal groups, ionizeto form a double ionized, through overall neutral entity identified aszwitterions. The amine donates an electron to the carboxyl group and theionic ends are stabilized in aqueous solution by polar water molecules.

As used herein, “AA” refers to an amino acid residue, i.e., the aminoacid without either a hydrogen atom or hydroxy group or amino group,depending on the linkage to the drug. For example, “OAA” refers to theamino acid forming an ester linkage with the oxygen atom of the drug.Thus, in this case, the amino acid is without a hydroxy group. However,the designation includes the ester being formed from the bond betweenthe carboxy group of the amino acid with the hydroxy group of the drug.However, if the amino acid has a carboxy group on the side chain, suchas glutamic acid or aspartic acid, this also refers to the ester beingformed from the carboxy group on the side chain and the hydroxy group ofthe drug. When aspartic or glutamic acid is the amino acid, the term OAArefers to an ester linkage between the drug and the amino acid from thecarboxy group on the main chain or on the side chain. Alternatively, OAArefers to the amino acid forming an ester linkage from the oxygen atomof the side chain of a hydroxy containing amino acid, and the carboxylicacid functionality of the drug. However, the definition should be madeclear by the context. For example, the nomenclature, especially in theclaims, is accompanied by language reciting that the bond is beingformed from a group, such as amino or OH group of the amino acid. Thus,for example, if the term is OAA, and if it is meant to refer to a esterbond between the OH group on the side chain and the acyl group of thedrug, the term would be accompanied by a statement that the amino acidis without the hydroxy group of the side chain and that the ester isformed via the hydroxy on the side chain. Alternatively, if the OAArefers to the ester linkage between the carboxy group of the amino acidand the oxygen atom of the drug, the term should be accompanied by astatement that the amino acid is without the hydroxy group and that theester linkage is through the acyl group of the amino acid. Further, ifthe structure of the drug to which the amino acid is bonded isidentified or provided, the meaning will be apparent to the skilledartisan without explanation.

The term NHAA refers to an amino acid residue (an amino acid less an OHgroup on the carboxy group) bonded through its acyl group to the aminogroup of the drug, thereby forming an amide linkage therebetween.Further, it should be noted that if the side chain of the amino acid hasa carboxy group thereon, another amide bond may occur linking thecarboxy group of the side chain and the amino group of the drug. Thus,the NHAA refers to the amide linkage between a carboxy group on theamino acid and the amino group on the drug. Thus, the term NHAA refersto the amide bond formed between the carboxy group of the drug and theamino group of the amino acid, either in the main chain or on the sidechain. Alternatively, it can refer to the amide linkage between theamino group of the amino acid and the carboxy group or acylatingderivative of the drug. Again, the term will be accompanied by languagein the claims to identify the meaning, if it is not clear from context.However, if the structure is given, the meaning will be apparent to theskilled artisan without explanation.

If there is a bond between the hydroxy group on the side chain of theamino acid, such as hydroxyproline, theonine, tyrosine, serine and thelike, then the nomenclature C(O)AA will be utilized, signifying that thelinkage is between the carboxy group on the drug and the OH group on theamino acid side chain.

The term

is used to denote that an acid anhydride is formed from the reaction ofthe carboxyl group of the drug and the carboxyl group of the amino acid,whether it be on the main, chain or side chain of the amino acid, toform an carboxylic acid anhydride.

The term —N═N-AA is used to denote the azo bond formed from the reactionof the amino group from the drug and the amino group of the amino acid.

It is the side groups that distinguish the amino acids from each other.Some amino acids, such as lysine, have amino groups on the side chain;other amino acids have side chains containing hydroxy groups, such asthreonine, serine, hydroxyproline, and tyrosine; some amino acids havecarboxy groups on the side chain, such as glutamic acid or asparticacid. These functional groups on the side chain also can form a covalentbond with the drug, such as esters, amides, and the like. When theseside groups become involved in these linkages, such as hydroxy group,the bond may be depicted as OAA, wherein AA is an amino acid residuehaving a side chain with a hydroxy group, but without the hydroxy group.Thus, AA by this definition, refers to the amino acid without thehydroxy side group since the hydroxy moiety took part in the reaction informing the ester. Moreover, when an ester is formed between the hydroxygroup of the amino acid and the carboxy group of the drug, the hydroxygroup on the carboxy group forms a byproduct with the hydrogen of thehydroxy group, thus, the resulting product does not have the OH group onthe carboxy group, but just the acyl moiety. When the bond is depictedas C(═O)—NHAA, this means that the amino acid forms as an amide bondbetween the carboxy group on the drug and the amino group of the aminoacid. However, as written, since the NH from the amide bond comes fromthe amino acid, AA is the amino acid without the amino group.

The preferred amino acids are the naturally occurring amino acids. It ismore preferred that the amino acids are the α-amino acids. It is alsopreferred that the amino acids are in the L-configuration. The preferredamino acids include the twenty essential amino acids. The preferredamino acids are Lysine (Lys), Leucine (Leu), Isoleucine (Ile), Glycine(Gly), Aspartic Acid (Asp), Glutamic Acid (Glu), Methionine (Met),Alanine (Ala), Valine (Val), Proline (Pro), Histidine (His), Tyrosine(Tyr), Serine (Ser), Norleucine (Nor), Arginine (Arg), Phenylalanine(Phe), Tryptophan (Trp), Hydroxyproline (Hyp), Homoserine (Hsr),Carnitine (Car), Ornithine (Ort), Canavanine (Cav), Asparagine (Asn),Glutamine (Gln), Carnosine (Can), Taurine (Tau), djenkolic Acid (Djk),γ-aminobutyric Acid (GABA), Cysteine (Cys) Cystine (Dcy), Sarcosine(Sar), Threonine (Thr) and the like. The even more preferred amino acidsare the twenty essential amino acids, Lys, Leu, Ile, Gly, Asp, Glu, Met,Ala, Val, Pro, His, Tyr, Thr, Arg, Phe, Trp, Gln, Asn, Cys and Ser. Themost preferred amino acids are those having a hydroxy group on the sidechain, preferably Hyp, Thr, Ser and Tyr and most preferably L-Hyp,L-Tyr, L-Ser and L-Thr. The most preferred amino acids are L-Hyp andL-Thr, and especially L-Thr.

The derivatives are prepared from a drug having a group thereon whichcan react with the amino acid.

The preferred drugs that are reacted with amino acids in accordance withvarious schemes are as follows. The drugs and their reaction schemesstated here are not exhaustive, and are typical examples only.

Reaction Schemes Drug A B C D E F Abacavir YES YES YES Acarbose YESAcebutolol YES YES Adefovir YES Albuterol YES YES Amlodipine¹ YESAmphotericin B YES YES YES YES Amprenavir YES YES YES Atenolol YES YESYES Atorvastatin YES YES YES YES Atropine YES Baclofen YES YES YES YESYES Benazeprilat YES YES YES YES Betaxolol YES YES Bicalutamide YES YESBiotin YES YES YES YES Biperiden YES Bisoprolol YES YES Bitolterol YESYES Brinzolamide YES YES Bupivacaine YES Buprenorphine YES Bupropion YESButorphanol YES Capacitabine YES Captopril YES YES YES YES Carbidopa YESYES YES YES YES YES Carnitine YES YES YES YES Carteolol YES YESCefditoren YES YES YES YES YES Cerivastatin YES YES YES YESChloramphenicol YES Cisapride YES YES Clopidogrel Acid YES YES YESClorazepic Acid YES YES YES YES Cycloserine YES YES Cytarabine YES YESYES Danazol YES Dextroamphetamine YES YES Diclofenac YES YES YES YESDidanosine YES YES Digoxin YES Divalproex YES YES YES Docetaxel YES YESDorzolamide YES YES Dyphylline YES Dysopyramide YES YES Efavirenz YESEnalaprilat YES YES YES YES Ephedrine YES YES Eplerenone YES YES YESEprosartan YES YES YES Esmolol YES YES Estramustine YES Ethambutol YESYES Ethchlorvynol YES Ethosuximide YES Ethotoin YES Etidocaine YESEtoposide YES Ezetimibe YES Fenofibrate YES YES YES Fenoprofen YES YESYES Fexofenadine YES YES YES YES Finasteride YES Fluoxetine YESFluticasone YES Fluvastatin YES YES YES YES Folic Acid YES YES YES YESYES Fosinoprilat YES YES Frovatriptan YES YES Fulvestrant YESGabapentin* YES YES YES YES YES Ganciclovir YES Glimepiride YESGoserelin YES Hydroxychloroquine YES Hydroxyzine YES Hyoscyamine YESIbuprofen YES YES YES Ibutilide YES YES Indapamide YES YES Indinavir YESYES Ipratropium YES Irinotecan YES Isosorbide YES Isradipine⁵ YESKetoprofen YES YES YES Ketorolac YES YES YES Labetalol YES YESLamivudine YES YES YES Lamivudine YES YES YES Lansoprazole YESLatanoprost Acid YES YES YES YES Leuprolide YES Levobunolol YES YES YESLevodopa YES YES YES YES YES YES Levorphanol YES Liothyronine YES YESYES YES YES YES Lisinopril YES YES YES YES YES Lopinavir YES YESLorazepam YES Lovastatin YES YES YES YES Medroxyprogesterone YESMefloquine YES YES Megestrol YES Mephobarbital YES Mepivacaine YESMetaproterenol YES YES Metformin YES YES Methamphetamine YESMethohexital YES Methotrexate YES YES YES Methylphenidate YES YES YESYES Methylphenidate⁶ YES Methylprednisolone YES Metolazone YES YESMetoprolol YES YES Mexiletine YES YES Miglitol YES Miglitol YESMoexiprilat YES YES YES YES Mometasone YES Montelukast YES YES YES YESNadolol YES YES Nalbuphine YES Naproxen YES YES YES Naratriptan YES YESNateglinide YES YES YES YES Nelfinavir YES YES Niacin YES YES YESNicardipine³ YES Nimidipine⁴ YES Nisoldipine² YES Norgestimate YESOctreotide YES YES Ofloxacin YES YES YES Olmesartan YES YES YESOmeprazole YES YES Paclitaxel YES YES Pantothenic Acid YES YES YES YESYES Paroxetine YES YES Paroxetine YES Pemoline YES YES Penbutolol YESYES Penicillamine YES YES YES YES YES Pentazocine YES Pentobarbital YESPerindoprilat YES YES YES YES Phenylephrine YES YES PhenylpropanolamineYES YES YES Pindolol YES YES Pioglitazone YES Pirbuterol YES PramipexoleYES YES Pravastatin YES YES YES YES Propafenone YES YES Propranolol YESYES Pseudoephedrine YES YES Quinacrine YES Quinaprilat YES YES YES YESQuinethazone YES YES Quinidine YES Quinine YES Ramiprilat YES YES YESYES Reboxetine YES Repaglinide YES YES YES YES Repaglinide YES YES YESYES YES Ribavirin YES YES YES Ritonavir YES YES Ropivacaine YESRosiglitazone YES Rosuvastatin YES YES YES YES Salmeterol YES YESSertraline YES Simavastatin YES YES YES YES Sirolimus YES Sotalol YESYES Sulfa Drugs YES YES Sulfasalazine YES Sumitriptan YES YES TacrolimusYES Tazorotene YES YES YES Telmesartan YES YES YES Tenofovir YESTerbutaline YES YES Thyroxine YES YES YES YES YES Tiagabine YES YES YESTimolol YES YES Tirofiban YES YES YES YES Tocainide YES YES Tramadol YESTrandolaprilat YES YES YES YES Tranylcypromine YES YES Treprostinil YESYES YES YES Triamcinolone YES Troglitazone YES YES Unoprostone YES YESYES Valsartan YES YES YES Venlafaxine YES Vidarabine YES YES YESWarfarin YES Zalcitabine YES YES YES Zidovudine YES YES Zolmitriptan YESYES ¹In case of Amlodipine, one can replace 5-methyl ester moiety withan amino acid resulting in better therapeutic index. In case of intactAmlodipine molecule, biotransformation results in generation of methanoldue to solvolysis of 5-methyl ester, which is highly toxic, andreplacement of this with a non-toxic naturally occurring amino acid withmuch less toxicity. In addition, the amino acid can form an amide bondwith the primary and secondary amine groups on the Amlodipine. Moreover,an amino acid can form an amide or ester bond at the 3 position of thering. This same argument goes for rest of the products in this categorystated below: ²In case of Nisoldipine, replace 5-methyl ester with anamino acid for better therapeutic index, and no loss of activity. Alsoan amide linkage may form between the amino group of the Nisoldipine andthe amino acid. In addition, an amide linkage or ester linkage can formbetween the 3 isobutyl ester and the amino acid. ³For Nicardipine,replace 5-methyl ester with an amino acid for better therapeutic index.In addition, the ester at the 3-position of the ring can form an esterlinkage with the OH group on the side chain. Further, the amino groupcan form an amide bond with a carboxy group or acylating derivativethereof of the amino acid. ⁴For Nimodipine, one could replace 5(1-methyl)ethyl ester with an amino acid for better therapeutic index.In addition, an amide can form between the amine group of the ring andthe amino acid. In addition, the 3 position of Nimodipine can form anamide bond or an ester bond with an amino acid. ⁵For Isradipine, replace5 methyl ester with an amino acid. The methyl ester is the active, andapparently the carboxylic acid derivative is not active. ⁶An amidelinkage can be formed between the secondary nitrogen atom in the ringand an amino acid. Moreover, the 3-position of the ring can form anamide or ester linkage with an amino acid. Replacing the methoxy groupwith amino acid will still maintain activity, but none of the toxicityof methylphenidate. In addition, an amide bond can form between thenitrogen atom of the pyridine and the amino acids.

The amino acid derivative of the present invention contains amino groupsand as such are basic in nature. They are capable of forming a widevariety of pharmaceutically acceptable salts with various inorganic andorganic acids. These acids that may be used to prepare pharmaceuticallyacceptable acid addition salts of such basic compounds are those thatform non-toxic acid addition salts, i.e., salts containingpharmaceutically acceptable anions, such as the hydrochloride,hydrobromide, hydroiodide, nitride, sulfate, bisulfate, phosphate,formate, acetate, citrate, tartate, lactate, and the like. The aminoacid derivative of the present invention can form pharmaceuticallyacceptable salts with acids.

As indicated herein, in one embodiment, the present invention isdirected to a derivative wherein the derivative comprises a drug, e.g.,cyclosporine of the formula

and an amino acid esterified to the MeBmt (x-y=CH═CH) or dihydro MeBmtportion, (wherein x-y=CH₂CH₂). The amino acid is attached to thecyclosporine and to the other drugs by a covalent bond.

The compounds of the present invention are prepared by art recognizedtechniques. For example, if the drug contains an OH group, said ascyclosporin, then an amino acid or an acylating derivatives thereof,such as the acid halide, e.g., amino acid fluoride, amino acid chloride,or an amino acid alkyl ester wherein alkyl group contains 1-6 carbonatoms is reacted with the carboxy group of the drug, e.g., cyclosporineunder esterification condition. Preferably, the reaction is conducted inthe presence of an acid, such as hydrochloric acid, hydrobromic acid,p-toluenesulfonic acid and the like. Alternatively, as describedhereinabove, if the drug has an amino group thereon, then the amino acidmay be reacted with the drug under amide forming conditions to form anamide as the covalent bond. Or if the drug has a carboxy group oracylating derivative thereon, it may be reacted with the amino group ofthe amino acid to form an amide under amide forming conditions to forman amide bond between the amino acid and the drug. Additionally if thedrug has a carboxy group thereon, the hydroxy group of the side chain ofthe amino acid may be reacted with the carboxy group or acylatingderivative thereon under esterification conditions to form the esterlinkage between the amino acid and the drug, as described hereinabove.

If the amino acid has a group thereon which is reactive under thereaction conditions it is protected by a protecting group known in theart. After the completion of the reaction, the protecting group isremoved. Examples of protecting groups that could be used are describedin the book entitled, “Protective Group in Organic Synthesis” byTheodora W. Greene, John Wiley & Sons, 1981, the contents of which areincorporated by reference.

For example, if amino acids with carboxylic groups in their side chains,for example, aspartic acid and glutamic acid, are used in theaforementioned syntheses, these will generally require protection of theside chain carboxylic acid. Suitable protecting groups can be esters,such as cyclohexyl esters, t-butyl esters, benzyl esters, allyl esters,9-fluorophenyl-methyl groups or adamantyl groups, such as 1- or2-adamantyl which can be removed after the esterfication reaction iscompleted using techniques known to one of ordinary skill in the art.

If amino acids with hydroxyl groups in their side chains, for example,serine, threonine, hydroxyproline, and the like and amino acids withphenolic groups in their side chains, for example, tyrosine, and thelike are used in the aforementioned esterification reactions, they willdesirably require protection of the chain hydroxyl or phenolic group.Suitable protecting groups for the hydroxyl side chain groups can beethers, such as benzyl ether or t-butyl ether and the like. Removal ofthe benzyl ether can be effected by liquid hydrogen fluoride, while thet-butyl ether can be removed by treatment with trifluoroacetic acid.Suitable protecting groups for the phenolic side chain groups can beethers, as above, including benzyl or t-butyl ether or2,6-dichlorobenzyl, 2-bromobenzyloxycarbonyl, 2,4-dintrophenyl and thelike.

Moreover, the products can be purified to be made substantially pure bytechniques known to one of ordinary skill in the art, such as bychromatography, e.g., HPLC, crystallization and the like. Bysubstantially “pure” it is meant that the product contains no more thanabout 10% impurity therein.

The amino acid derivatives of the present invention encompasspharmaceutical acceptable salts, pharmaceutical acceptable solvates,enantiomers, diastereomers, N-Oxides, and polymorphs thereof, asdescribed herein, and they can be associated along with a pharmaceuticalacceptable carrier, and optionally but desirably pharmaceuticallyacceptable excipients and made into pharmaceutical composition usingtechniques known to one of ordinary skill in the art.

All the various stereoisomers of the amino acid derivatives, such asenantiomers and dieastercomers are comtemplated to be within the scopeof the present invention. However, if the preferred drug has a preferredsteroisomeric form, it is also preferred in the present invention.Moreover, the preferred amino acid derivatives of the present inventionhas the asymmetric carbon atom on the group adjacent to the amino andcarboxy group from the amino acid moiety in the L configuration.

The amino acid derivatives described herein have the same utility asthat drug without the amino acid moiety bonded thereto. The amino acidderivatives are used in therapeutically effective amounts.

The physician will determine the dosage of the derivatives of thepresent invention which will be most suitable and it will vary with theform of administration and the particular compound chosen, andfurthermore, it will vary depending upon various factors, including butnot limited to the patient under treatment and the age of the patient,the severity of the condition being treated and the like and theidentify of the derivative administered. He will generally wish toinitiate treatment with small dosages, substantially less than theoptimum dose of the compound, and increase the dosage by smallincrements until the optimum effect under the circumstances is reached.It will generally be found that when the composition is administeredorally, larger quantities of the compounds of the present invention willbe required to produce the same effect as a smaller quantity givenparenterally. The amino acid derivatives of the present invention havethe same utility as the corresponding drug in the non amino acidderivatized form and the dosage level is generally no greater than thatis generally employed with these other therapeutic agents. When givenparenterally, the compounds are administered generally in dosages of,for example, about 0.001 to about 10,000 mg/kg/day, also depending uponthe host and the severity of the condition being treated and thecompound utilized.

In a preferred embodiment, the compounds of the present inventionutilized are orally administered in amounts ranging from about 0.01 mgto about 1000 mg per kilogram of body weight per day, depending upon theparticular mammalian host or the disease to be treated, more preferablyfrom about 0.1 to about 500 mg/kg body weight per day. This dosageregimen may be adjusted by the physician to provide the optimumtherapeutic response. For example, several divided doses may beadministered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

The amino acid derivative of the present invention may be administeredin any convenient manner, such as by oral, intravenous, intramuscular orsubcutaneous routes.

The amino acid derivative of the present invention may be orallyadministered, for example, with an inert diluent or with an assimilableedible carrier, or it may be enclosed in hard or soft shell gelatincapsules, or it may be compressed into tablets, or it may beincorporated directly into the food of the diet. For oral therapeuticadministration, the amino acid derivative of the present invention maybe incorporated with excipients and used in the form of ingestibletablets, buccal tablets, troches, capsules, elixirs, suspensions,syrups, wafers, and the like. Such compositions and preparations shouldcontain at least 1% of the derivative. The percentage of thecompositions and preparations may, of course, be varied and mayconveniently be between about 5 to about 80% of the weight of the unit.The amount of the amino acid derivative used in such therapeuticcompositions is such that a suitable dosage will be obtained. Preferredcompositions or preparations according to the present invention containbetween about 200 mg and about 4000 mg of amino acid derivative. Thetablets, troches, pills, capsules and the like may also contain thefollowing: A binder such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, lactose or saccharin may be added or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier.

Various other materials may be present as coatings or otherwise modifythe physical form of the dosage unit. For instance, tablets, pills, orcapsules may be coated with shellac, sugar or both. A syrup or elixirmay contain the active compound, sucrose as a sweetening agent, methyland propylparabens as preservatives, a dye and flavoring such as cherryor orange flavor. Of course, any material used in preparing any dosageunit form should be pharmaceutically pure and substantially non-toxic inthe amounts employed. In addition, the active compound may beincorporated into sustained-release preparations and formulations. Forexample, sustained release dosage forms are contemplated wherein theactive ingredient is bound to an ion exchange resin which, optionally,can be coated with a diffusion barrier coating to modify the releaseproperties of the resin or wherein the derivative of the presentinvention is associated with a sustained release polymer known in theart, such as hydroxypropylmethylcellulose and the like.

The amino acid derivative may also be administered parenterally orintraperitoneally. It is especially advantageous to formulate parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dispersions can also be prepared in glycerol,liquid polyethylene glycols, e.g., PEG 100, PEG 200, PEG 300, PEG 400,and the like, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. In all cases the form is usually sterile andmust be fluid to the extent that syringability exists. It must be stableunder the conditions of manufacture and storage and usually must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (for example, glycerol,propylene glycol, and one or more liquid polyethylene glycol, e.g. asdisclosed herein and the like), suitable mixtures thereof, and vegetableoils. The proper fluidity can be maintained, for example, by the use ofa coating such as lecithin, by the maintenance of the required particlesize in the case of dispersions and by the use of surfactants. Theprevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

Sterile injectable solutions are prepared by incorporating the aminoacid derivative of the present invention in the required amount in theappropriate solvent with various of the other ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the various sterilized aminoacid derivative of the present invention into a sterile vehicle whichcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders, the abovesolutions are vacuum dried or freeze-dried, as necessary.

The amino acid derivative of the present invention can also be appliedtopically, as e.g., through a patch using techniques known to one ofordinary skill in the art.

The amino acid derivative of the present invention can be administeredbuccally by preparing a suitable formulation of the derivative of thepresent invention and utilizing procedures well known to those skilledin the art. These formulations are prepared with suitable non-toxicpharmaceutically acceptable ingredients. These ingredients are known tothose skilled in the preparation of buccal dosage forms. Some of theseingredients can be found in Remington's Pharmaceutical Sciences, 17^(th)edition, 1985, a standard reference in the field. The choice of suitablecarriers is highly dependent upon the exact nature of the buccal dosageform desired, e.g., tablets, lozenges, gels, patches and the like. Allof these buccal dosage forms are contemplated to be within the scope ofthe present invention and they are formulated in a conventional manner.

The formulation of the pharmaceutical compositions may be prepared usingconventional methods using one or more physiologically and/orpharmaceutically acceptable carriers or excipients. Thus, the amino acidderivatives and their pharmaceutically acceptable salts and solvates maybe formulated for administration by inhalation or insufflation (eitherthrough the mouth or the nose) or oral, buccal, parenteral, or rectaladministration. For oral administration, the pharmaceutical compositionsmay take the form of, for example, tablets or capsules prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (for example, pregelatinized maize starch,polyvinylpyrrolidone, or hydroxypropylmethyl cellulose); fillers (forexample, lactose, microcrystalline cellulose or calcium hydrogenphosphate); lubricants (for example, magnesium stearate, talc, orsilica); disintegrants (for example, potato starch, or sodium starchglycolate); or wetting agents (for example, sodium lauryl sulfate). Thetablets may be coated by methods well known in the art.

Liquid preparations for oral administration may take the form of, forexample, solutions, syrups, or suspensions, or they may be presented asa dry product for constitution with water or other suitable vehiclesbefore use. Such liquid preparations may be prepared by conventionalmeans with pharmaceutically acceptable additives, such as suspendingagents (for example, sorbitol syrup, corn syrup, cellulose derivativesor hydrogenated edible oils and fats); emulsifying agents (for example,lecithin or acacia); non-aqueous vehicles (for example, almond oil, oilyesters, ethyl alcohol or fractionated vegetable oils); and preservatives(for example, methyl or propyl p-hydroxybenzoates or sorbic acid). Thepreparations may also contain buffer salts, flavoring, coloring andsweetening agents as appropriate. Preparations for oral administrationmay be suitably formulated to give controlled release of the amino acidderivative of the present invention.

The amino acid derivative of the present invention may be formulated forparenteral administration by injection, for example, by bolus injectionor continuous infusion. Formulations for injection may be presented inunit dosage form, for example, in ampoules, or in multi-dose containers,with an added preservative. The compositions may take such forms assuspension, solutions or emulsions in oily or aqueous vehicles, and maycontain formulation agents such as suspending, stabilizing and/ordispersing agents. Alternatively, the amino acid derivative may be inthe powder form for constitution with a suitable vehicle, for example,sterile pyrogen-free water, before use.

The amino acid derivatives of the present invention may also beformulated in rectal compositions such as suppositories or retentionenemas, for example, containing conventional suppository bases such ascocoa butter or other glycerides.

In addition to the formulations described previously, the amino acidderivative of the present invention may also be formulated as a depotpreparation. Such long acting formulations may be administered byimplantation (for example, subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, the amino acid derivativesmay be formulated with suitable polymeric or hydrophobic materials (forexample, as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

The pharmaceutical compositions containing the amino acid derivatives ofthe present invention may, if desired, be presented in a pack ordispenser device which may contain one or more unit dosage formscontaining the active ingredients. The pack may for example comprisemetal or plastic foil, such as blister pack. The pack or dispenserdevice may be accompanied by instructions for administration.

In tablet form, it is desirable to include a lubricant which facilitatesthe process of manufacturing the dosage units; lubricants may alsooptimize erosion rate and drug flux. If a lubricant is present, it willbe present on the order of 0.01 wt. % to about 2 wt. %, preferably about0.01 wt. % to 0.5 wt, %, of the dosage unit. Suitable lubricantsinclude, but are not limited to, magnesium stearate, calcium stearate,stearic acid, sodium stearylfumarate, talc, hydrogenated vegetable oilsand polyethylene glycol. As will be appreciated by those skilled in theart, however, modulating the particle size of the components in thedosage unit and/or the density of the unit can provide a similareffect—i.e., improved manufacturability and optimization of erosion rateand drug flux—without addition of a lubricant.

Other components may also optionally be incorporated into the dosageunit. Such additional optional components include, for example, one ormore disintegrants, diluents, binders, enhancers, or the like. Examplesof disintegrants that may be used include, but are not limited to,crosslinked polyvinylpyrrolidones, such as crospovidone (e.g.,Polyplasdone® XL, which may be obtained from GAF), cross-linkedcarboxylic methylcelluloses, such as croscanmelose (e.g., Ac-di-sol®,which may be obtained from FMC), alginic acid, and sodium carboxymethylstarches (e.g., Explotab®, which may be obtained from Edward Medell Co.,Inc.), agar bentonite and alginic acid. Suitable diluents are thosewhich are generally useful in pharmaceutical formulations prepared usingcompression techniques, e.g., dicalcium phosphate dihydrate (e.g.,Di-Tab®, which may be obtained from Stauffer), sugars that have beenprocessed by crystallization with dextrin (e.g., co-crystallized sucroseand dextrin such as Di-Pak®, which may be obtained from Amstar), calciumphosphate, cellulose, kaolin, mannitol, sodium chloride, dry starch,powdered sugar and the like. Binders, if used, are those that enhanceadhesion. Examples of such binders include, but are not limited to,starch, gelatin and sugars such as sucrose, dextrose, molasses, andlactose. Permeation enhancers may also be present in the novel dosageunits in order to increase the rate at which the active agents passthrough the buccal mucosa. Examples of permeation enhancers include, butare not limited to, dimethylsulfoxide (“DMSO”), dimethyl formamide(“DMF”), N,N-dimethylacetamide (“DMA”), decylmethylsulfoxide (“C₁₀MSO”),polyethylene glycol monolaurate (“PEGML”), glycerol monolaurate,lecithin, the 1-substituted azacycloheptan-2-ones, particularly1-n-dodecylcyclazacycloheptan-2-one (available under the trademarkAzone® from Nelson Research & Development Co., Irvine, Calif.), loweralkanols (e.g., ethanol), SEPA® (available from Macrochem Co.,Lexington, Mass.), cholic acid, taurocholic acid, bile salt typeenhancers, and surfactants such as Tergitol®, Nonoxynol-9® andTWEEN-80®.

Flavorings may be optionally included in the various pharmaceuticalformulations. Any suitable flavoring may be used, e.g., mannitol,lactose or artificial sweeteners such as aspartame. Coloring agents maybe added, although again, such agents are not required. Examples ofcoloring agents include any of the water soluble FD&C dyes, mixtures ofthe same, or their corresponding lakes.

In addition, if desired, the present dosage units may be formulated withone or more preservatives or bacteriostatic agents, e.g., methylhydroxybenzoate, propyl hydroxybenzoate, chlorocresol, benzalkoniumchloride, or the like.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents well known in the artthat are associated with drugs, medicaments, or active agents. Exceptinsofar as any conventional media or agent is incompatible with theamino acid derivative, the use of these solvents, dispersion media,coatings and various isotonic and delaying agents and antibacterical andantifungal agents in the therapeutic compositions of the presentinvention is contemplated. Supplementary active ingredients can also beincorporated into the compositions.

Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated, each unitcontaining a predetermined quantity of amino acid derivative calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier.

The amino acid derivative is compounded for convenient and effectiveadministration in effective amounts with a suitable pharmaceuticallyacceptable carrier in dosage unit form as hereinbefore described. A unitdosage, for example, contains the amino acid derivative of the presentinvention in amounts ranging from about 10 mg e.g. in humans, or as lowas 1 mg (for small animals) to about 2000 mg. If placed in solution, theconcentration of the amino acids derivative preferably ranges from about10 mg/mL to about 250 mg/mL. In the case of compositions containingsupplementary active ingredients, the dosages are determined byreference to the usual dose and manner of administration of the saidingredients. In the case of buccal administration, the amino acidderivatives are preferably in the buccal unit dosage form present in anamount ranging from about 10 to about 50 mg.

The amino acid derivatives of the present invention are effective intreating disease or conditions in which the corresponding drug (withoutthe amino acid derivative of the present invention) normally are used.

As used herein the term “treating” refers to reversing, alleviating orinhibiting the progress of a disease, disorder or condition, or one ormore symptoms of such disease, disorder or condition, to which such termapplies. As used herein, “treating” may also refer to decreasing theprobability or incidence of the occurrence of a disease, disorder orcondition in a mammal as compared to an untreated control population, oras compared to the same mammal prior to treatment. For example, as usedherein, “treating” may refer to preventing a disease, disorder orcondition, and may include delaying or preventing the onset of adisease, disorder or condition, or delaying or preventing the symptomsassociated with a disease, disorder or condition. As used herein,“treating” may also refer to reducing the severity of a disease,disorder or condition or symptoms associated with such disease, disorderor condition prior to a mammal's affliction with the disease, disorderor condition. Such prevention or reduction of the severity of a disease,disorder or condition prior to affliction relates to the administrationof the composition of the present invention, as described herein, to asubject that is not at the time of administration afflicted with thedisease, disorder or condition. As used herein “treating” may also referto preventing the recurrence of a disease, disorder or condition or ofone or more symptoms associated with such disease, disorder orcondition. The terms “treatment” and “therapeutically,” as used herein,refer to the act of treating, as “treating” is defined above.

Prophylaxis or preventing, or any like term, refers to decreasing theprobability or incidence of the occurrence of a disease, disorder orcondition in a mammal. It also includes delaying or preventing the onsetof a disease, disorder or condition or delaying the symptoms associatedwith a disease, disorder or condition. In addition, it also refers toretarding the occurrence of a disease, disorder or condition in amammal.

As used herein the term “patient” or “subject” refers to a warm bloodedanimal, and preferably mammals, such as, for example, cats, dogs,horses, cows, pigs, mice, rats and primates, including humans. Thepreferred patient is humans.

The amino acid derivatives of the present invention exhibit the sameutility as the corresponding drug without the amino acid linkage. Theamino acid derivative of the present invention exhibits an enhancedtherapeutic quality. That is, they exhibit at least one and morepreferably at least two enhanced therapeutic qualities relative to thedrug which has not been transformed to the derivative of the presentinvention prior to administration. These include, but are not limited to

-   -   a. Improved taste, smell    -   b. Desired octanol/water partition coefficient (i.e., solubility        in water/fat)

The various amino acids have different solubility in aqueous solutions.By selecting a particular amino acid, the octanol water partitioncoefficient can be affected. For example, many drugs in the followinglist are highly hydrophobic. The amino acids are highly hydrophilic. Forexample, assume propofol is the drug and lysine is the amino acid.Propofol is completely insoluble in water, while lysine is soluble tothe extent of 700 mg/ml. When these two diverse molecules are esterifiedvia an ester bond, the resulting lysine ester of propfol has asolubility in water in excess of 250 mg/ml.

On the other hand, cromolyn sodium is highly water soluble. For allpractical purposes, it is not absorbed when administered orally. Byaffecting its water solubility one could improve absorption. In thiscase, one would look for conditions opposite to that of propofol, i.e.,the goal is to decrease water solubility. By choosing the appropriatelow water soluble amino acids, such as tyrosine, one can achieve properhydrophilic/lipophilic balance.

-   -   c. Improved stability in-vitro and in-vivo    -   d. Enhanced penetration of blood-brain barrier    -   e. Elimination of first-pass effect in liver, i.e., the drug not        metabolized in liver and therefore more drug in system        circulation    -   f. Reduction of entero-hepatic recirculation (this improves        bio-availability)    -   g. Painless injections with parenteral formulations    -   h. Improved bio-availability    -   i. Improved changes in the rate of absorption (increase vs lack        thereof)    -   j. Reduced side effects    -   k. Dose proportionality

A dose proportionality claim requires that when the drug is administeredin escalating doses, proportionally escalating amounts of active drug isdelivered into the blood stream. This is measured by determining thearea under the plasma concentration vs. time curve obtained afteradministering a drug via any route other than IV route and measuring thesame in the plasma/blood. A simple mathematical procedure is as follows:

For example, a drug is administered at e.g., 3 different doses, 10, 100and 1000 mg, orally to a patient; the area under the plasmaconcentration time curve (AUC) is measured. Then each total AUC isdivided by the dose, and the result should be the same for all threedoses. If it is the case, then there is dose proportionality. Lack ofdose proportionality indicates any one or more of thepharmacokinetic/pharmacological mechanisms are saturable, includingabsorption, metabolism or the number of receptor sites available forpharmacological response.

For example in the above study, assume the AUC values of 100, 1000 and10,000 are obtained, in this case the dose proportionality isinappropriate. When there is lack of dose proportionality, there iseither more or less amount of drug in the plasma, depending upon whichmechanism is saturable. The following are the possibilities: SaturableAbsorption. If this is the case, as the dose is increased,proportionally less and less of the drug is absorbed, hence overall AUCwill decrease as the dose is increased.

Saturable metabolism of elimination. If thus is the case, then more andmore of the drug will be circulating in the blood, and the AUC willincrease with increasing dose.

Saturable pharmacological receptor sites: In this case, since all thereceptor sites will eventually be occupied by the drug, any additionaldrug will not increase the response. Thus, increasing dose will notresult in increasing response.

Dose proportionality is an excellent response profile, since one canpredict accurately the pharmacological response and curative power atall doses. Thus dose proportionality is a desirable quality for anydrug. Furthermore, achievement of dose proportionality is also dependentupon the formulation, and fed/fasted differences.

-   -   l. Selective hydrolysis of the derivative at site of action    -   m. Controlled release properties    -   n. Targeted drug delivery    -   o. Reduction in toxicity, hence, improved therapeutic ratio    -   p. Reduced dose    -   q. Alteration of metabolic pathway to deliver more drug at the        site of action    -   r. Increased solubility in aqueous solution    -   s. Enhanced efficacy

The amino acid derivatives are available in various dosage forms andthey are prepared by conventional methods:

-   -   i. Oral liquid dosage (Controlled release and immediate release        liquids containing sugar and sugar free, dye and dye free,        alcohol and alcohol free formulations, including chewable        tablets)    -   ii. Oral solid dosage (Controlled release and immediate release        tablets, capsules and caplets    -   iii. Intravenous (Injections, both ready to use and lyophilized        powders)    -   iv. Intramuscular (Injections, both ready to use and lyophilized        powders)    -   v. Subcutaneous (Injections, both ready to use and lyophilized        powders)    -   vi. Transdermal (Mainly patches)    -   vii. Nasal (Sprays, formulations for nebulizer treatments)    -   viii. Topical (Creams, ointments)    -   ix. Rectal (Creams, ointments and suppositories)    -   x. Vaginal (Creams, ointments and pessaries)    -   xi. Ocular (Drops and ointments)    -   xii. Buccal (Chewable and now chewable tables)

Many drugs discussed herein, especially in the table hereinbelow arecharacteristically highly hydrophobic and readily precipitate in thepresence of even very minor amounts of water, e.g., on contact with thebody (e.g., stomach fluids). It is accordingly extremely difficult toprovide, e.g., oral formulations which are acceptable to the patient interms of form and taste, which are stable on storage and which can beadministered on a regular basis to provide suitable and controllingpatient dosing.

Proposed liquid formulations, e.g., for oral administration of a numberof drugs shown herein in the table have heretofore been based primarilyon the use of ethanol and oils or similar excipient as carrier media.Thus, the commercially available drink-solutions of a number of drugsemploy ethanol and olive oil or corn oil as carrier medium inconjunction with solvent systems comprising e.g., ethanol and LABRIFILand equivalent excipient as carrier media. For example, the commerciallyavailable Cyclosporin drink solution employs ethanol and olive oil orcorn oil as carrier medium in conjunctions with a Labroid as asurfactant. See e.g., U.S. Pat. No. 4,388,307. Use of the drink solutionand similar composition as proposed in the art is however accompanied bya variety of difficulties.

Further, the palatability of the known oil based system has provedproblematic. The taste of the known drink-solution of several drugs is,in particular, unpleasant. Admixture with an appropriate flavored drink,for example, chocolate drink preparation, at high dilution immediatelyprior to ingestion has generally been practiced in order to make regulartherapy at all acceptable. Adoption of oil-based systems has alsorequired the use of high ethanol concentrations which is itselfinherently undesirable, in particular where administration to childrenis foreseen. In addition, evaporation of the ethanol, e.g., fromcapsules (adopted in large part, to meet problems of palatability, asdiscussed or other forms (e.g., when opened)) results in the developmentof a drug precipitate.

Where such compositions are presented in, for example, soft gelatinencapsulated form, this particular difficulty necessitates packaging ofthe encapsulated product in an air-tight component, for example, anair-tight blister or aluminum-foil blister package. This in turn rendersthe product both bulky and more expensive to produce. The storagecharacteristics of the aforesaid formulations are, in addition, far fromideal.

Bioavailability levels achieved using existing oral dosage system for anumber of drugs described herein are also low and exhibit wide variationbetween individuals, individual patient types and even for singleindividuals at different times during the course of therapy. Reports inthe literature indicate that currently available therapy employing thecommercially available drug drink solution provides an average absolutebioavailability of approximately 10-30% only, with a marked variationbetween individual groups, e.g., between liver (relatively lowbioavailability) and bone-marrow (relatively high bioavailability)transplant recipients. Reported variation in bioavailability betweensubjects has varied from one or a few percent for some patients, to asmuch as 90% or more for others. And as already noted, marked change inbioavailability for individuals with time is frequently observed. Thus,there is a need for a more uniform and high bioavailability of a numberdrugs in patients.

Use of dosage forms of existing drugs is also characterized by extremevariation in required patient dosing. To achieve effective therapy, drugblood or blood serum levels have to be maintained within a specifiedrange. This required range can in turn, vary, depending on theparticular condition being treated, e.g., whether therapy is to preventone or more pharmacological actions of a specific drug and whenalternative therapy is employed concomitantly with principal therapy.Because of the wide variations in bioavailability levels achieved withconventional dosage forms, daily dosages needed to achieve requiredblood serum levels will also vary considerably from individual toindividual and even for a single individual. For this reason it may benecessary to monitor blood/blood-serum levels of patients receiving drugtherapy at regular and frequent intervals. Monitoring ofblood/blood-serum levels has to be carried out on a regular basis. Thisis inevitably time consuming and inconvenient and adds substantially tothe overall cost of therapy.

It is also the case that blood/blood serum levels of a number of drugswithout the amino acid linkage described herein achieved using availabledosage systems exhibit extreme variation between peak and trough levels.That is, for each patient, effective drug levels in the blood varywidely between administrations of individual dosages.

There is also a need for providing a number of drugs described herein,especially the beta-lactum antibiotics, Cyclosporin, cephalosporins,steroids, quinolone antibiotics and the like, in a water-soluble formfor injection. It is well known that Cremaphore L® (CreL) used incurrent formulations of a number of drugs described hereinbelow is apolyoxyethylated derivative of castor oil and is a toxic vehicle. Therehave been a number of incidences of anaphylaxis due to the castor oilcomponent. At present there is no formulation that would allow many ofthese drugs to be in aqueous solution at the concentrations needed dueto poor water solubility of the drug.

Beyond all these very evident practical difficulties lies the occurrenceof undesirable side reactions already alluded to, observed employingavailable oral dosage forms.

Several proposals to meet these various problems have been suggested inthe art, including both solid and liquid oral dosage forms. Anoverriding difficulty which has however remained is the inherentinsolubility of the several of the drugs without the amino acid linkageshown in the table hereinbelow in aqueous media, hence preventing theuse of a dosage form which can contain the drugs in sufficiently highconcentration to permit convenient use and yet meet the requiredcriteria in terms of bioavailability, e.g. enabling effective absorptionfrom the stomach or gut lumen and achievement of consistent andappropriately high blood/blood-serum levels, is needed.

The particular difficulties encountered in relation to oral dosing withthese drugs have inevitably led to restrictions in the use of specificdrug therapy for the treatment of relatively less severe or endangeringdisease conditions. For example, taking Cyclosporin as a test drug, aparticular area of difficulty in this respect has been the adoption ofCyclosporin therapy in the treatment of autoimmune diseases and otherconditions affecting the skin, for example for the treatment of atopicdermatitis and psoriasis and, as also widely proposed in the art, forhair growth stimulation, e.g. in the treatment of alopecia due to ageingor disease.

Thus while oral Cyclosporin therapy has shown that the drug is ofconsiderable potential benefit to patients suffering e.g. frompsoriasis, the risk of side-reaction following oral therapy hasprevented common use. Various proposals have been made in the art forapplication of Cyclosporins, e.g. Cyclosporin, in topical form and anumber of topical delivery systems have been described. Attempts attopical application have however failed to provide any demonstrablyeffective therapy.

However, the present invention overcomes the problems describedhereinabove. More specifically, the amino acid derivative of the presentinvention significantly enhances its solubility in aqueous solutionsrelative to the non-derivative form of the pharmaceutical, therebyavoiding the need to utilize a carrier, such as ethanol or castor oilwhen administered as a solution. Moreover, the amino acid derivatives ofthese drugs, in accordance with the present invention, do not exhibitthe side effects of the prior art formulations. Further, it has beenfound that when many of the drugs in the table hereinbelow isadministered in its amino acid derivative form in accordance with thepresent invention, there is enhanced oral absorption, thereby enhancingsignificantly its bioavailability and its efficacy.

The preferred drugs used in combination with the amino acids formingderivatives are listed hereinbelow in the following table and thebenefits found are as listed in the penultimate column of the table. Inthe table, the key is as follows:

-   -   a) Improved taste smell    -   b) Desired Octanol/water partition coefficient (i.e. solubility        in water)    -   c) Improved stability in vitro and in vivo    -   d) Penetration of blood-brain barrier    -   e) Elimination of first pass effect in liver    -   f) Reduction of enterohepatic recirculation    -   g) Painless injections with parenteral formulations    -   h) Improved bioavailability    -   i) Increased rate of absorption    -   j) Reduced side effects    -   k) Dose proportionability    -   l) Selective hydrolysis of the derivative at site of actions    -   m) Controlled release properties    -   n) Targeted drug delivery    -   o) Reduction in toxicity, hence improved therapeutic ratio    -   p) Reduced dose    -   q) Alteration of metabolic pathway to deliver more drug at site        of action.

Moreover, the table indicates the utility of the derivative. The utilityof the derivative is the same as the corresponding drug (without theamino acid moiety attached). The utility is described in the literaturesuch as in the Physicians Desk Reference, 2004 edition, the contents ofwhich are incorporated by reference.

TABLE 1 Amino Acids that can Improvements Applicable Preferred Most bereacted with the Most with Dose Dose Preferred drug to form thePreferred derivatives Amino Acid Range Range Dose Rangester/amide/azo/anhydride Preferred Amino utility Derivatives of Alldoses expressed as drug base derivatives Amino Acids Acids immunoUtility Cyclosporin 5-1000 mg 20-250 mg 25-100 mg Lys, Leu, Ile, Gly,Lys, Leu, Ile, Gly, Lys, Pro, b, e, f, g, h, prophylaxis of organPreferred 1-25 mg/ml 5-15 mg/ml 10 mg/ml Asp, Glu, Met, Ala, Asp, Glu,Met, Ala, & Gly and k, l, o, and p rejection, e.g., kidney, Forms 10-250mg 25-100 mg 50 mg/5 ml Val, Pro, His, Tyr, Ser, Val, Pro, His, Tyr,dipeptides liver and heart allogenic Oral Tab/Cap per 5 ml per 5 ml Nor,Arg, Phe, Trp, Thr, Arg, Phe, Trp, of Lys- transplants, treatment ofOral Liquid Hyp, Hsr, Car, Ort, Gln, Asn, Cys and Gly, Pro- rheumatoidarthritis and IV Injections Cav, Asn, Gln, Can, Ser, Hyp, Sar or Gly,Gly- psoriosis Tau, Djk, GABA, Cys, dipeptides of Gly Dcy, Thr, and Saror combination of any dipeptide of two amino acids combination of anyespecially AA-Gly, two amino acids where Gly is a especially AA-Gly,spacer attached to where Gly is a spacer cyclosporin and attached to AAis the above- cyclosporin and AA is cited amino acids. the above-citedamino acids. Lopinavir 0.1-1 gm 200-800 mg 400-500 mg Lys, Leu, Ile,Gly, Lys, Leu, Ile, Gly, Lys, Pro, b, h, j, k, treatment of HIVPreferred 0.1-1 gm/5 ml 0.2-0.8 g/ 400 mg/5 ml Asp, Glu, Met, Ala, Asp,Glu, Met, Ala, Gly, & and o infections, e.g., AIDS Forms 5 ml Val, Pro,His, Tyr, Ser, Val, Pro, His, Tyr, Ala Oral Tab/Cap Nor, Arg, Phe, Trp,Thr, Arg, Phe, Trp, Oral Liquid Hyp, Hsr, Car, Ort, Gln, Asn, Cys andCav, Asn, Gln, Can, Ser, Hyp, Sar Tau, Djk, GABA, Cys, Dcy, Thr, and SarCefdinir 0.1-1 gm 0.2-0.5 gm 200-400 mg Lys, Leu, Ile, Gly, Lys, Leu,Ile, Gly, Ser, a, b, e, f, h, antibiotic treatment of diseases causedPreferred 0.1-1 gm/ 0.2-0.5 gm/ 0.2-0.4 gm/ Asp, Glu, Met, Ala, Asp,Glu, Met, Ala, Hyp, i, o, and p by Haemophilus influenzae, includingForms 5 ml 5 ml 5 ml Val, Pro, His, Tyr, Ser, Val, Pro, His, Tyr, Tyr, &B-lactamase producing strains, e.g., Oral Tab/Cap 0.01-1 gm/ 20-500 mg/50-150 mg/ Nor, Arg, Phe, Trp, Thr, Arg, Phe, Trp, Thr Haemophilusparainfluenzae (including Oral Liquid 100 ml 100 ml 100 ml Hyp, Hsr,Car, Ort, Gln, Asn, Cys and β-lactosamase producing strains) and IVInfusions Cav, Asn, Gln, Can, Ser, Hyp, Sar moraxella catarihalis(including β- Tau, Djk, GABA, Cys, lactamase producing strains), andDcy, Thr, and Sar streptococcus pyogenes; such as pneumonia, bronchitisand sinusitis, pharyngitis and tonsillitis Zileuton 200-1200 mg 200-800mg 300-400 mg Lys, Leu, Ile, Gly, Lys, Leu, Ile, Gly, Gly, Lys, b, h, i,j, k, treatment of asthma Preferred 200-1200 mg/ 200-800 mg/ 200-400 mg/Asp, Glu, Met, Ala, Asp, Glu, Met, Ala, Sar, Ala, o, p Forms 5 ml 5 ml 5ml Val, Pro, His, Tyr, Ser, Val, Pro, His, Tyr, Pro Oral Tab/Cap Nor,Arg, Phe, Trp, Thr, Arg, Phe, Trp, Oral Liquid Hyp, Hsr, Car, Ort, Gln,Asn, Cys and Cav, Asn, Gln, Can, Ser, Hyp, Sar Tau, Djk, GABA, Cys, Dcy,Thr, and Sar Nelfinavir 0.05-1 gm 0.1-0.5 gm 0.2-0.4 gm Lys, Leu, Ile,Gly, Lys, Leu, Ile, Gly, Gly, Lys, b, h, i, j, k, treatment of HIV,infected patients, Preferred 10-250 mg/gm 20-200 mg/gm 40-100 mg/gm Asp,Glu, Met, Ala, Asp, Glu, Met, Ala, Sar, Ala, o, p e.g., AIDS forms10-250 mg/ 20-200 mg/ 40-100 mg/ Val, Pro, His, Tyr, Ser, Val, Pro, His,Tyr, Pro Oral Tab/Cap 100 ml 100 ml 100 ml Nor, Arg, Phe, Trp, Thr, Arg,Phe, Trp, Oral Powder Hyp, Hsr, Car, Ort, Gln, Asn, Cys and IV Cav, Asn,Gln, Can, Ser, Hyp, Sar Formulation Tau, Djk, GABA, Cys, Dcy, Thr, andSar Flavoxate 10-1000 mg 20-500 mg 50-250 mg Lys, Leu, Ile, Gly, Asp,Lys, Leu, Ile, Gly, Hyp, Ser, Tyr, b, h, i, j, k, treatment of urinaryspasms Preferred Forms 10-1000 mg/ 20-500 mg/ 50-250 mg/ Glu, Met, Ala,Val, Pro, Asp, Glu, Met, Ala, & Thr l, o, & p Oral Tab/Cap 5 ml 5 ml 5ml His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, Oral Liquid Phe, Trp,Hyp, Hsr, Thr, Arg, Phe, Trp, Car, Ort, Cav, Asn, Gln, Asn, Cys and Gln,Can, Tau, Djk, Ser, Hyp, Sar GABA, Cys, Dcy, Thr, and Sar Candesarten1-100 mg 2-75 mg 4-50 Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Hyp,Ser, Tyr, b, c, e, f, treatment of hypertension Preferred Forms 1-100mg/ 2-75 mg/ 4-50 mg/ Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, & Thrh, i, j, k, l, Oral Tab/Cap 5 ml 5 ml 5 ml His, Tyr, Ser, Nor, Arg, Val,Pro, His, Tyr, o, p, q Oral Liquid Phe, Trp, Hyp, Hsr, Thr, Arg, Phe,Trp, Car, Ort, Cav, Asn, Gln, Asn, Cys and Gln, Can, Tau, Djk, Ser, Hyp,Sar GABA, Cys, Dcy, Thr, and Sar Propofol 1-25 mg/ml 2.0-20 mg/ml 5-15mg/ml Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Gly, Lys, Sar, b, c,d, g, provides central nervous Preferred Forms Glu, Met, Ala, Val, Pro,Asp, Glu, Met, Ala, Pro, Ala, & h, j, k, l, system anesthesia IVInfusions His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, Val m, n, o, p, qPhe, Trp, Hyp, Hsr, Thr, Arg, Phe, Trp, Car, Ort, Cav, Asn, Gln, Asn,Cys and Gln, Can, Tau, Djk, Ser, Hyp, Sar GABA, Cys, Dcy, Thr, and SarNisoldipine 2-100 mg 2.5-75 mg 5-50 mg Lys, Leu, Ile, Gly, Asp, Lys,Leu, Ile, Gly, Gly, Lys, Ser, b, e, h, i, j, o calcium channel blocker,Preferred Forms 2-100 mg/ 2.5-75 mg/ 5-50 mg/ Glu, Met, Ala, Val, Pro,Asp, Glu, Met, Ala, & Hyp treatment of hypertension Oral Tab/Cap 5 ml 5ml 5 ml His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, Oral Liquid Phe,Trp, Hyp, Hsr, Thr, Arg, Phe, Trp, Car, Ort, Cav, Asn, Gln, Asn, Cys andGln, Can, Tau, Djk, Ser, Hyp, Sar GABA, Cys, Dcy, Thr, and SarAmlodipine 0.1-20 mg 1-10 mg 2.5-5 mg Lys, Leu, Ile, Gly, Lys, Leu, Ile,Gly, Gly, Lys, Ser, b, e, h, i, j, o calcium channel blocker, Preferredforms IV 0.1-20 mg/ 1-10 mg/ 2.5-5 mg/ Asp, Glu, Met, Ala, Asp, Glu,Met, Ala, & Hyp treatment of hypertension Oral Tab/Cap 5 ml 5 ml 5 mlVal, Pro, His, Tyr, Ser, Val, Pro, His, Tyr, Oral Liquid Nor, Arg, Phe,Trp, Thr, Arg, Phe, Trp, Hyp, Hsr, Car, Ort, Gln, Asn, Cys and Cav, Asn,Gln, Can, Ser, Hyp, Sar Tau, Djk, GABA, Cys, Dcy, Thr, and SarCiprofloxacin 0.1-1.5 gm 0.1-1.0 gm 0.2-0.8 gm Lys, Leu, Ile, Gly, Lys,Leu, Ile, Gly, Hyp, Ser, a, b, c, g, Antibiotic; inhibits variousPreferred Forms 0.05-1 gm/ 0.08-1 gm/ 0.12-1 g/5 ml Asp, Glu, Met, Ala,Asp, Glu, Met, Ala, Thr, Gly, & h, i, j, k, o, p bacteria, e.g.,pseudomonas Oral Tab/Cap 5 ml 5 ml 5-15 mg/ml Val, Pro, His, Tyr, Ser,Val, Pro, His, Tyr, Lys aeruginosa, staphylococcus Oral Liquid 2-25mg/ml 3-20 mg/ml Nor, Arg, Phe, Trp, Thr, Arg, Phe, Trp, aureus orproteus mirabilis; IV Bulk (Sterile) Hyp, Hsr, Car, Ort, Gln, Asn, Cysand treatment of corneal ulcers, Cav, Asn, Gln, Can, Ser, Hyp, Sarconjunctivitis, acute otitis Tau, Djk, GABA, Cys, externa, Dcy, Thr, andSar Ramipril 0.1-20 mg 0.5-12 mg 1-10 mg Lys, Leu, Ile, Gly, Lys, Leu,Ile, Gly, Hyp, Ser, j, o treatment of hypertension Preferred Forms0.1-20 mg/ 0.5-12 mg/ 1-10 mg/5 ml Asp, Glu, Met, Ala, Asp, Glu, Met,Ala, Thr, Gly, & Oral Tab/Cap 5 ml 5 ml Val, Pro, His, Tyr, Ser, Val,Pro, His, Tyr, Lys Oral Liquid Nor, Arg, Phe, Trp, Thr, Arg, Phe, Trp,Hyp, Hsr, Car, Ort, Gln, Asn, Cys and Cav, Asn, Gln, Can, Ser, Hyp, SarTau, Djk, GABA, Cys, Dcy, Thr, and Sar Trandolapril 0.1-10 mg 0.5-7.5 mg1-4 mg Lys, Leu, Ile, Gly, Lys, Leu, Ile, Gly, Ser, Hyp, j, o treatmentof hypertension Preferred Forms 0.1-10 mg/ 0.5-7.5 mg/ 1-4 mg/5 ml Asp,Glu, Met, Ala, Asp, Glu, Met, Ala, Thr, Gly & Oral Tab/Cap 5 ml 5 mlVal, Pro, His, Tyr, Ser, Val, Pro, His, Tyr, Lys Oral Liquid Nor, Arg,Phe, Trp, Thr, Arg, Phe, Trp, Hyp, Hsr, Car, Ort, Gln, Asn, Cys and Cav,Asn, Gln, Can, Ser, Hyp, Sar Tau, Djk, GABA, Cys, Dcy, Thr, and SarFosinopril 1-100 mg 2-75 mg 5-50 mg Lys, Leu, Ile, Gly, Lys, Leu, Ile,Gly, Ser, Hyp, j, o treatment of hypertension Preferred Forms 1-100 mg/2-75 mg/5 ml 5-50 mg/ Asp, Glu, Met, Ala, Asp, Glu, Met, Ala, Thr, Gly,& Oral Tab/Cap 5 ml 5 ml Val, Pro, His, Tyr, Ser, Val, Pro, His, Tyr,Lys Oral Liquid Nor, Arg, Phe, Trp, Thr, Arg, Phe, Trp, Hyp, Hsr, Car,Ort, Gln, Asn, Cys and Cav, Asn, Gln, Can, Ser, Hyp, Sar Tau, Djk, GABA,Cys, Dcy, Thr, and Sar Enalapril 0.5-100 mg 1-50 mg 2-25 mg Lys, Leu,Ile, Gly, Lys, Leu, Ile, Gly, Ser, Hyp, j, o treatment of hypertensionPreferred forms 0.5-100 mg/ 1-50 mg/5 ml 2-25 mg/ Asp, Glu, Met, Ala,Asp, Glu, Met, Ala, Thr, Gly & Oral Tab/Cap 5 ml 5 ml Val, Pro, His,Tyr, Ser, Val, Pro, His, Tyr, Lys Oral Liquid Nor, Arg, Phe, Trp, Thr,Arg, Phe, Trp, Hyp, Hsr, Car, Ort, Gln, Asn, Cys and Cav, Asn, Gln, Can,Ser, Hyp, Sar Tau, Djk, GABA, Cys, Dcy, Thr, and Sar Benazepril 1-100 mg2-75 mg 2.5-50 mg Lys, Leu, Ile, Gly, Lys, Leu, Ile, Gly, Hyp, Ser, j, otreatment of hypertension Preferred Forms 1-100 mg/ 2-75 mg/5 ml 2.5-50mg/ Asp, Glu, Met, Ala, Asp, Glu, Met, Ala, Thr, Gly, & Oral Tab/Cap 5ml 5 ml Val, Pro, His, Tyr, Ser, Val, Pro, His, Tyr, Lys Oral LiquidNor, Arg, Phe, Trp, Thr, Arg, Phe, Trp, Hyp, Hsr, Car, Ort, Gln, Asn,Cys and Cav, Asn, Gln, Can, Ser, Hyp, Sar Tau, Djk, GABA, Cys, Dcy, Thr,and Sar Perindopril 0.1-20 mg 0.5-15 mg 1-10 mg Lys, Leu, Ile, Gly, Lys,Leu, Ile, Gly, Hyp, Ser, j, o treatment of hypertension Preferred Forms0.1-20 mg/ 0.5-15 mg/5 ml 1-10 mg/ Asp, Glu, Met, Ala, Asp, Glu, Met,Ala, Thr, Gly, & Oral Tab/Cap 5 ml 5 ml Val, Pro, His, Tyr, Ser, Val,Pro, His, Tyr, Lys Oral Liquid Nor, Arg, Phe, Trp, Thr, Arg, Phe, Trp,Hyp, Hsr, Car, Ort, Gln, Asn, Cys and Cav, Asn, Gln, Can, Ser, Hyp, SarTau, Djk, GABA, Cys, Dcy, Thr, and Sar Moexipril 1-30 mg 2-20 mg 5-15 mgLys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Ser, Hyp, Thr, j, otreatment of hypertension Preferred Forms 1-30 mg/5 ml 2-20 mg/5 ml 5-15mg/ Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Gly & Lys Oral Tab/Cap5 ml His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, Oral Liquid Phe, Trp,Hyp, Hsr, Thr, Arg, Phe, Trp, Car, Ort, Cav, Asn, Gln, Asn, Cys and Gln,Can, Tau, Djk, Ser, Hyp, Sar GABA, Cys, Dcy, Thr, and Sar Cromolyn10-200 mg 20-100 mg 20-50 mg Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile,Gly, Ser, Hyp, Thr, & b, c, h, i, j, inhibits release of histaminePreferred Forms 10-200 mg/ 20-100 mg/ 20-50 mg/ Glu, Met, Ala, Val, Pro,Asp, Glu, Met, Ala, Pro k, l, n, o, and leukotrienes from mast OralTab/Cap 5 ml 5 ml 5 ml His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, p, qcell; treatment of Oral Liquid Phe, Trp, Hyp, Hsr, Thr, Arg, Phe, Trp,mastocytosis, asthma Car, Ort, Cav, Asn, Gln, Asn, Cys and Gln, Can,Tau, Djk, Ser, Hyp, Sar GABA, Cys, Dcy, Thr, and Sar Amoxicillin 0.1-1.5gm 0.2-1.2 gm 0.25-1 gm Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly,Ser, Hyp, Thr, a, b, c, h, antibiotic effective against Preferred forms0.1-1.5 gm/ 0.2-1.2 gm/ 0.25-1 gm/ Glu, Met, Ala, Val, Pro, Asp, Glu,Met, Ala, Gly & Lys i, j, k, l, o, p β-lactamase negative strains OralTab/Cap* 5 ml 5 ml 5 ml His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr,causing infections of ear, Oral Liquid 0.1-0.75 gm 0.1-0.6 gm 0.125-0.5gm Phe, Trp, Hyp, Hsr, Thr, Arg, Phe, Trp, nose, throat, e.g., OralPowder Car, Ort, Cav, Asn, Gln, Asn, Cys and streptococcus, (* alsochewable) Gln, Can, Tau, Djk, Ser, Hyp, Sar staphylococcus or H GABA,Cys, Dcy, Thr, influenzae; treatment of and Sar infections ofgenitourinary tract due to E. coli, P. mirabilis, E. faecalis,infections of skin due to streptococcus, staphylococcus or E. coli,infections of lower respiratory tract due to streptoccus, staphyloccusor H. influenzae, and gonorrhea Cefuroxime 10-1000 mg 50-750 mg 100-600mg Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Hyp, Ser, Thr, b, c, e,f, antibiotic; treatment of Preferred Forms 10-1000 mg/ 50-750 mg/100-600 mg/ Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Gly, & Lys h,i, j, k, o, p pharyngitis/tonsillitis caused Oral Tab/Cap 5 ml 5 ml 5 mlHis, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, by streptococcus, acuteOral Liquid Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp, bacterialotitismedia caused Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and bystreptococcus, H Tau, Djk, GABA, Cys, Ser, Hyp, Sar influenzae,moraxella Dcy, Thr, and Sar catarihalis or streptococcus, urinary tractinfections caused by E. coli or Klebsiella pneumonia, gonorrhea, skininfections cause by staphylococcus or straptococcus Ceftazidime 0.1-5 gm0.25-4 gm 0.5-2 gm Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Hyp,Ser, Thr, a, b, c, g, antibiotic, treatmtent of Preferred Forms 0.1-1 gm0.25-1 gm 0.5-1 gm Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Gly, &Lys h, i, j, k, l, lower respiratory tract Powder for IV 0.1-2.5 gm/0.25-2 gm/ 0.5-1 gm/ His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, o, p,q infections, including Oral Tab/Cap 5 ml 5 ml 5 ml Phe, Trp, Hyp, Hsr,Car, Thr, Arg, Phe, Trp, pneumonia caused by Oral Liquid Ort, Cav, Asn,Gln, Can, Gln, Asn, Cys and pseudomonas, H. influenzae, Tau, Djk, GABA,Cys, Ser, Hyp, Sar Klebsiella, Dcy, Thr, and Sar Enterbacter, E. coli,proteus mirabilis, streptococcus, staphylococcus; skin and skinstructure infections, caused by pseudomonas aeruginosa, Klebsiella, E.coli, Proteus enterbacter, staphylococcus, streptococcus, urinary tractinfections casued by pseudomonas aeruginosa, enterbacter, proteus,Klebsiella, E. coli; bone and joint infections caused by pseudomonas,eruginosa, Klebsiella, Enterbacter, or staphylococcus; gynecologicinfections including endometritis, pelvic cellulits and infections ofthe female genital tract caused by E. coli, intra-abdominal infectionsand central nervous system infections, including meningitis Cefpodoxime10-500 mg 25-350 mg 50-250 mg Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile,Gly, Ser, Hyp, Thr, a, b, c, g, h, i, antibiotic especially PreferredForms 10-500 mg/ 25-350 mg/ 50-250 mg/ Glu, Met, Ala, Val, Pro, Asp,Glu, Met, Ala, Gly & Lys j, k, l, o, p, q against streptococcus, OralTab/Cap 5 ml 5 ml 5 ml His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, H.influenzae, Oral Liquid Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp,moraxella catarrhalis; Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys andtreatment of acute otis Tau, Djk, GABA, Cys, Ser, Hyp, Sar media,pharyngitis, Dcy, Thr, and Sar tonsillitis, pneumonia, bronchitis,gonorrhea, and rectal infections in women Atovaquone 50-1000 mg 100-500mg 200-300 mg Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Lys, Gly,Sar, a, b, h, i, j, k, treatment of malaria Preferred Forms above/5 mlabove/5 ml above/5 ml Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Ala,Pro & Ser o, p caused by plasmodium Oral Tab/Cap 10-150 mg/ 25-100 mg/50-75 mg/ His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, parasite OralLiquid 5 ml 5 ml 5 ml Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp, ForPediatric Use Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and Tau, Djk, GABA,Cys, Ser, Hyp, Sar Dcy, Thr, and Sar Acyclovir 50-1000 mg 100-750 ml150-500 mg Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Lys, Sar, Hyp b,c, h, i, j, k, treatment of human Preferred forms 50-1000 mg/ 100-750mg/ 150-500 mg/ Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Pro & Sero, p cytomegalovirus Oral Tab/Cap 5 ml 5 ml 5 ml His, Tyr, Ser, Nor,Arg, Val, Pro, His, Tyr, (HCMV) Oral Liquid Phe, Trp, Hyp, Hsr, Car,Thr, Arg, Phe, Trp, Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and Tau, Djk,GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and Sar Gancyclovir 0.1-1 gm 0.2-0.8gm 0.2-0.6 gm Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Hyp, Ser,Thr, b, c, e, f, h, i, j, treatment of human Preferred Forms 0.1-1 gm/5ml 0.2-0.8 mg/ 0.2-0.6 mg/ Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala,Gly, & Lys k, o, p cytomegalo virus Oral Tab/Cap 10-200 mg/ml 5 ml 5 mlHis, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, (HCMV) Oral Liquid 25-100mg/ml 30-60 mg/ml Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp, IVInfusions Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and Tau, Djk, GABA,Cys, Ser, Hyp, Sar Dcy, Thr, and Sar Penciclovir 10-1000 mg/ml 25-750mg/ml 50-500 mg/ml Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Hyp,Ser, Thr, b, c, e, f, h, i, j, treatment of human Preferred Forms 0.1-5%0.25-3% 0.5-2.5% Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Gly, & Lysk, o, p cytomegalovirus Powder for IV 10-500 mg 20-300 mg 25-250 mg His,Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, (HCMV) Topical Cream Phe, Trp,Hyp, Hsr, Car, Thr, Arg, Phe, Trp, Oral Cap/Tab Ort, Cav, Asn, Gln, Can,Gln, Asn, Cys and Tau, Djk, GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and SarNiacin ER 0.2-2 gm 0.25-1.5 gm 0.5-1 gm Lys, Leu, Ile, Gly, Asp, Lys,Leu, Ile, Gly, Ser, Hyp, Thr, a, b, h, i, j, l, lipid managementPreferred Forms 0.2-2 gm/5 ml 0.25-1 gm/ 0.5-1 gm/ Glu, Met, Ala, Val,Pro, Asp, Glu, Met, Ala, Tyr, Gly & Lys m, n, o, p, q Oral Tab/Cap 5 ml5 ml His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, Oral Liquid Phe, Trp,Hyp, Hsr, Car, Thr, Arg, Phe, Trp, Ort, Cav, Asn, Gln, Can, Gln, Asn,Cys and Tau, Djk, GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and Sar Bexarotene10-500 mg 25-250 mg 50-100 mg Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile,Gly, Ser, Hyp, Thr, b, c, h, i, j, k, l, treatment of skin PreferredForms above/5 ml above/5 m above/5 ml Glu, Met, Ala, Val, Pro, Asp, Glu,Met, Ala, Gly, & Lys o, p conditions, especially Oral Tab/Cap 0.1-5%0.25-2.5% l 0.5-1.5% His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, thoserequiring Oral Liquid Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp,activation of retinoid Topical Gel Ort, Cav, Asn, Gln, Can, Gln, Asn,Cys and X receptors Tau, Djk, GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and SarPropoxyphene 20-400 mg 25-250 mg 30-150 mg Lys, Leu, Ile, Gly, Asp, Lys,Leu, Ile, Gly, Ser, Hyp, Thr, a, b, c, h, i, j, treatment of painPreferred forms 20-400 mg/ 25-250 mg/ 30-150 mg/ Glu, Met, Ala, Val,Pro, Asp, Glu, Met, Ala, Gly, & Lys k, l, o, p Oral Tab/Cap 5 ml 5 ml 5ml His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, Oral Liquid Phe, Trp,Hyp, Hsr, Car, Thr, Arg, Phe, Trp, Ort, Cav, Asn, Gln, Can, Gln, Asn,Cys and Tau, Djk, GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and Sar Salsalate0.2-2 gm 0.25-1.5 gm 0.3-1 gm Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile,Gly, Hyp, Ser, Thr, Gly, b, c, h, i, j, treatment of Preferred Forms0.2-2 gm/5 ml 0.25-1.5 gm/ 0.3-1 gm/ Glu, Met, Ala, Val, Pro, Asp, Glu,Met, Ala, & Lys k, o, p inflammatory Oral Tab/Cap 5 ml 5 ml His, Tyr,Ser, Nor, Arg, Val, Pro, His, Tyr, conditions Oral Liquid Phe, Trp, Hyp,Hsr, Car, Thr, Arg, Phe, Trp, Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys andTau, Djk, GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and Sar Acetaminophen20-1000 mg 50-800 mg 100-600 mg Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile,Gly, Hyp, Ser, Sar, Gly, a, b, c, e, h, treatment of pain or PreferredForms 20-1000 mg/ 50-800 mg/ 100-600 mg/ Glu, Met, Ala, Val, Pro, Asp,Glu, Met, Ala, & Lys i, j, k, o, p fever Oral Tab/Cap 5 ml 5 ml 5 mlHis, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, Oral Liquid Phe, Trp, Hyp,Hsr, Car, Thr, Arg, Phe, Trp, Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys andTau, Djk, GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and Sar Ibuprofen 20-1000mg 50-800 mg 100-600 mg Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly,Ser, Hyp, Thr, Tyr, a, b, h, i, j, l, treatment of pain, fever PreferredForms 20-1000 mg/ 50-800 mg/ 100-600 mg/ Glu, Met, Ala, Val, Pro, Asp,Glu, Met, Ala, Gly & Lys m, n, o, p, q or inflammation Oral Tab/Cap 5 ml5 ml 5 ml His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, Oral Liquid Phe,Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp, Ort, Cav, Asn, Gln, Can, Gln,Asn, Cys and Tau, Djk, GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and SarLovastatin 1-100 mg 2-80 mg 5-50 mg Lys, Leu, Ile, Gly, Asp, Lys, Leu,Ile, Gly, Ser, Hyp, Thr, Gly, b, c, e, f, lowers choloesterol PreferredForms 1-100 mg/5 ml 2-80 mg/ 5-50 mg/ Glu, Met, Ala, Val, Pro, Asp, Glu,Met, Ala, & Lys h, i, j, k, concentration; Oral Tab/Cap 5 ml 5 ml His,Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, l, o, p inhibits HMG-CoA OralLiquid Phe, Trp, Hyp, Hsr, Thr, Arg, Phe, Trp, reductase Car, Ort, Cav,Asn, Gln, Asn, Cys and Gln, Can, Tau, Djk, Ser, Hyp, Sar GABA, Cys, Dcy,Thr, and Sar Simavastatin 1-200 mg 2-150 mg 2.5-100 mg Lys, Leu, Ile,Gly, Asp, Lys, Leu, Ile, Gly, Ser, Hyp, Thr, Gly, b, c, e, f, lowerscholoesterol Preferred forms 1-200 mg/5 ml 2-150 mg/ 2.5-100 mg/ Glu,Met, Ala, Val, Pro, Asp, Glu, Met, Ala, & Lys h, i, j, k, concentration;Oral Tab/Cap 5 ml 5 ml His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, l,o, p inhibits HMG-CoA Oral Liquid Phe, Trp, Hyp, Hsr, Thr, Arg, Phe,Trp, reductase Car, Ort, Cav, Asn, Gln, Asn, Cys and Gln, Can, Tau, Djk,Ser, Hyp, Sar GABA, Cys, Dcy, Thr, and Sar Atorvastatin 1-250 mg 2-125mg 5-100 mg Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Ser, Hyp, Thr,Gly, b, c, e, f, lowers choloesterol Preferred Forms 1-250 mg/5 ml 2-125mg/ 5-100 mg/ Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, & Lys h, i,j, k, concentration; Oral Tab/Cap 5 ml 5 ml His, Tyr, Ser, Nor, Arg,Val, Pro, His, Tyr, l, o, p inhibits HMG-CoA Oral Liquid Phe, Trp, Hyp,Hsr, Thr, Arg, Phe, Trp, reductase Car, Ort, Cav, Asn, Gln, Asn, Cys andGln, Can, Tau, Djk, Ser, Hyp, Sar GABA, Cys, Dcy, Thr, and SarPravastatin 1-250 mg 2-125 mg 5-75 mg Lys, Leu, Ile, Gly, Asp, Lys, Leu,Ile, Gly, Ser, Hyp, Thr, Gly, b, c, e, f, lowers choloesterol PreferredForms 1-250 mg/5 ml 2-125 mg/ 5-75 mg/ Glu, Met, Ala, Val, Pro, Asp,Glu, Met, Ala, & Lys h, i, j, k, concentration; Oral Tab/Cap 5 ml 5 mlHis, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, l, o, p inhibits HMG-CoAOral Liquid Phe, Trp, Hyp, Hsr, Thr, Arg, Phe, Trp, reductase Car, Ort,Cav, Asn, Gln, Asn, Cys and Gln, Can, Tau, Djk, Ser, Hyp, Sar GABA, Cys,Dcy, Thr, and Sar Fluvastatin 1-250 mg 2-125 mg 5-75 mg Lys, Leu, Ile,Gly, Asp, Lys, Leu, Ile, Gly, Ser, Hyp, Thr, Tyr, b, c, e, f, lowerscholoesterol Preferred Forms 1-250 mg/5 ml 2-125 mg/ 5-75 mg/ Glu, Met,Ala, Val, Pro, Asp, Glu, Met, Ala, Gly & Lys h, i, j, k, concentration;Oral Tab/Cap 5 ml 5 ml His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, l,o, p inhibits HMG-CoA Oral Liquid Phe, Trp, Hyp, Hsr, Thr, Arg, Phe,Trp, reductase Car, Ort, Cav, Asn, Gln, Asn, Cys and Gln, Can, Tau, Djk,Ser, Hyp, Sar GABA, Cys, Dcy, Thr, and Sar Nadolol 1-250 mg 5-225 mg10-200 mg Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Lys, Gly, Sar,Ser, b, h, i, j, treatment of angina Preferred Forms 1-250 mg/5 ml 5-225mg/ 10-200 mg/ Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, & Pro k, l,o, p pectoris and Oral Tab/Cap 5 ml 5 ml His, Tyr, Ser, Nor, Arg, Val,Pro, His, Tyr, hypertension; β- Oral Liquid Phe, Trp, Hyp, Hsr, Thr,Arg, Phe, Trp, adrenergic receptor Car, Ort, Cav, Asn, Gln, Asn, Cys andantagonist Gln, Can, Tau, Djk, Ser, Hyp, Sar GABA, Cys, Dcy, Thr, andSar Valsartan 10-500 mg 25-250 mg 50-200 mg Lys, Leu, Ile, Gly, Asp,Lys, Leu, Ile, Gly, Hyp, Ser, Thr, Lys, b, f, i, j, treatinghypertension, Preferred forms 10-500 mg/ 25-250 mg/ 50-200 mg/ Glu, Met,Ala, Val, Pro, Asp, Glu, Met, Ala, Gly & Sar k, l, o, p angiotension IIOral Tab/Cap 5 ml 5 ml 5 ml His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr,antagonist Oral Liquid Phe, Trp, Hyp, Hsr, Thr, Arg, Phe, Trp, Car, Ort,Cav, Asn, Gln, Asn, Cys and Gln, Can, Tau, Djk, Ser, Hyp, Sar GABA, Cys,Dcy, Thr, and Sar Methyl 1-50 mg 2-40 mg 2.5-25 mg Lys, Leu, Ile, Gly,Asp, Lys, Leu, Ile, Gly, Lys, Gly, Hyp, Sar, a, b, c, h, treatment ofattention phenidate 1-50 mg/5 ml 2-40 mg/ 2.5-25 mg/5 ml Glu, Met, Ala,Val, Pro, Asp, Glu, Met, Ala, & Ser j, k, l, o, p deficit disorders andPreferred Forms 5 ml His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr,narcolepsy Oral Tab/Cap Phe, Trp, Hyp, Hsr, Thr, Arg, Phe, Trp, OralLiquid Car, Ort, Cav, Asn, Gln, Asn, Cys and Gln, Can, Tau, Djk, Ser,Hyp, Sar GABA, Cys, Dcy, Thr, and Sar Trovafloxacin 10-500 mg 50-300 mg80-250 mg Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Lys, Gly, Ser,Pro, a, b, e, h, j, antibiotic; inhibits Preferred Forms 10-500 mg/50-300 mg/ 80-250 mg/ Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Hyp &Thr k, o, p bacteria such as E. coli, Oral Tab/Cap 5 ml 5 ml 5 ml His,Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, pseudomonas, Oral Liquid Phe,Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp, aeruginosa, H. influenzae, Ort,Cav, Asn, Gln, Can, Gln, Asn, Cys and streptococous, Tau, Djk, GABA,Cys, Ser, Hyp, Sar Klebsiella, Dcy, Thr, and Sar staphylococcus,mycoplasma pneumoniae, peptostreptococcus, prevotella; treatment ofpneumonia, postsurgical infections; gynecolgic and pelvic infections,such as endomyometritis, parametritis, septic abortions, and post-partum infections; skin infections, e.g., diabetic foot infections 5-AS*1-200 mg 5-150 mg 10-125 mg Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly,Glu, Gly, Tyr & Lys b, c, i, j, l, treatment of Preferred Forms 1-200mg/5 ml 5-150 mg/ 10-125 mg/ Glu, Met, Ala, Val, Pro, Asp, Glu, Met,Ala, m, n, o, p, q tuberculosis Oral Tab/Cap 5 ml 5 ml His, Tyr, Ser,Nor, Arg, Val, Pro, His, Tyr, Oral Liquid Phe, Trp, Hyp, Hsr, Car, Thr,Arg, Phe, Trp, (* 5-Amino- Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys andSalicylic acid) Tau, Djk, GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and SarMethyl 2-200 mg/ml 5-150 mg/ml 10-100 mg/ml Lys, Leu, Ile, Gly, Asp,Lys, Leu, Ile, Gly, Lys, Gly, Pro, b, c, g, j, l, treatment ofprednisolone 0.001-5% 0.01-2.5% 0.1-2% Glu, Met, Ala, Val, Pro, Asp,Glu, Met, Ala, Sar & Ser m, n, o, p, q inflammation Preferred Forms His,Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, especially from IM InjectionPhe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp, infections, tissue TopicalCream Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and damage, allergy andTau, Djk, GABA, Cys, Ser, Hyp, Sar auto-immune Dcy, Thr, and Sar diseaseMedroxy 1 mg-4 gm/ml 10 mg-2 gm/ml 40 mg-1 gm/ml Lys, Leu, Ile, Gly,Asp, Lys, Leu, Ile, Gly, Lys, Gly, Pro, b, c, g, j, l, providingProgesterone Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Sar & Ser m,n, o, p, q contraception Preferred forms His, Tyr, Ser, Nor, Arg, Val,Pro, His, Tyr, IM Injections Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe,Trp, Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and Tau, Djk, GABA, Cys,Ser, Hyp, Sar Dcy, Thr, and Sar Estramustine 10-500 mg 25-250 mg 50-200mg Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Gly, Lys, Pro, Ala, b,c, h, i, j, treatment of cancer Preferred Forms 10-500 mg/ 25-250 mg/50-200 mg/ Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Sar & Val k, l,o, p especially treatment Oral Tab/Cap 5 ml 5 ml 5 ml His, Tyr, Ser,Nor, Arg, Val, Pro, His, Tyr, of metastatic or Oral Liquid Phe, Trp,Hyp, Hsr, Car, Thr, Arg, Phe, Trp, progressive Ort, Cav, Asn, Gln, Can,Gln, Asn, Cys and carcinoma of Tau, Djk, GABA, Cys, Ser, Hyp, Sarprostate Dcy, Thr, and Sar Miglitol 1-250 mg 2-150 mg 10-125 mg Lys,Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Lys, Gly, Sar, Pro, & b, c, i,j, n, q treatment of type II Preferred Forms 1-250 mg/5 ml 2-150 mg/10-125 mg/ Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Ser diabetesOral Tab/Cap 5 ml 5 ml His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, OralLiquid Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp, Ort, Cav, Asn, Gln,Can, Gln, Asn, Cys and Tau, Djk, GABA, Cys, Ser, Hyp, Sar Dcy, Thr, andSar Mefloquine 10-500 mg 100-400 mg 150-300 mg Lys, Leu, Ile, Gly, Asp,Lys, Leu, Ile, Gly, Lys, Gly, Sar, Pro, a, b, c, h, i, treatment ofmalaria Preferred Forms 10-500 mg/ 25-400 mg/ 150-300 mg/ Glu, Met, Ala,Val, Pro, Asp, Glu, Met, Ala, Val, Ala j, k, l, o, p, q Oral Tab/Cap 5ml 5 ml 5 ml His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, Oral LiquidPhe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp, Ort, Cav, Asn, Gln, Can,Gln, Asn, Cys and Tau, Djk, GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and SarDanazol 2-500 mg 10-350 mg 25-250 mg Lys, Leu, Ile, Gly, Asp, Lys, Leu,Ile, Gly, Hyp, Pro, Ala, Val, a, b, c, e, f, treatment of Preferredforms 2-500 mg/ 10-350 mg/ 25-250 mg/ Glu, Met, Ala, Val, Pro, Asp, Glu,Met, Ala, Ser & Thr g, h, i, j, k, endometriosis and Oral Tab/Cap 5 ml 5ml 5 ml His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, l, n, o, p, qfibrostatic breast Oral Liquid Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe,Trp, disease Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and Tau, Djk, GABA,Cys, Ser, Hyp, Sar Dcy, Thr, and Sar Eprosartan 0.1-1 gm 200-800 mg300-750 mg Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Ser, Hyp, Thr,Lys, b, c, h, i, j, ACE inhibitors, Preferred Forms 0.1-1 gm/ 200-800mg/ 300-750 mg/ Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Gly & Valk, l, o, p treatment of Oral Tab/Cap 5 ml 5 ml 5 ml His, Tyr, Ser, Nor,Arg, Val, Pro, His, Tyr, hypertension Oral Liquid Phe, Trp, Hyp, Hsr,Car, Thr, Arg, Phe, Trp, Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and Tau,Djk, GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and Sar Divalproex Na 50-800 mg75-750 mg 100-600 mg Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Ser,Hyp, Thr, Lys, a, b, c, f, h, treatment of epilepsy Preferred Forms50-800 mg/ 75-750 mg/ 100-60 mg/ Glu, Met, Ala, Val, Pro, Asp, Glu, Met,Ala, Gly, & Val i, j, k, l, o, p Oral Tab/Cap 5 ml 5 ml 5 ml His, Tyr,Ser, Nor, Arg, Val, Pro, His, Tyr, Oral Liquid Phe, Trp, Hyp, Hsr, Car,Thr, Arg, Phe, Trp, Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and Tau, Djk,GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and Sar Fenofibrate 10-800 mg 20-750mg 100-600 mg Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Ser, Hyp,Thr, Lys, b, c, h, i, j, treatment of Preferred Forms 10-800 mg/ 20-750mg/ 100-600 mg/ Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Gly, & Alak, l, o, p, q hypercholestemia Oral Tab/Cap 5 ml 5 ml 5 ml His, Tyr,Ser, Nor, Arg, Val, Pro, His, Tyr, Oral Liquid Phe, Trp, Hyp, Hsr, Car,Thr, Arg, Phe, Trp, Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and Tau, Djk,GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and Sar Gabapentin 10-800 mg 25-750mg 50-500 mg Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Cyclic Deriv.& Tyr b, c, d, e, f, treatment of Preferred Forms 10-800 mg/ 25-750 mg/50-500 mg/ Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, h, i, j, k, l,convulsions Oral Tab/Cap 5 ml 5 ml 5 ml His, Tyr, Ser, Nor, Arg, Val,Pro, His, Tyr, n, o, p, p Oral Liquid Phe, Trp, Hyp, Hsr, Car, Thr, Arg,Phe, Trp, Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and Tau, Djk, GABA,Cys, Ser, Hyp, Sar Dcy, Thr, and Sar Lansoprazole 1-60 mg 2-50 mg 10-40mg Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Gly, Lys, Pro, Sar, b,e, f, h, i, suppression of gastric Preferred Forms 1-6-mg/ 2-50 mg/5 ml10-40 mg/ Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Ser & Val j, k,l, o, p acid secretion by Oral Tab/Cap 5 ml 5 ml His, Tyr, Ser, Nor,Arg, Val, Pro, His, Tyr, inhibition of (H+, K+) Oral Liquid Phe, Trp,Hyp, Hsr, Car, Thr, Arg, Phe, Trp, ATP-ase enzyme Ort, Cav, Asn, Gln,Can, Gln, Asn, Cys and system at the secretory Tau, Djk, GABA, Cys, Ser,Hyp, Sar surface of the gastric Dcy, Thr, and Sar parietal cell;treatment of gastric hyperacidity Omeprazole 1-200 mg 2-100 mg 5-60 mgLys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Lys, Gly, Val, Pro & b, e,f, h, i, suppression of gastric Preferred Forms 1-200 mg/ 2-100 mg/ 5-60mg/ Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Sar j, k, l, o, p acidsecretion by Oral Tab/Cap 5 ml 5 ml 5 ml His, Tyr, Ser, Nor, Arg, Val,Pro, His, Tyr, inhibition of (H+, K+) Oral Liquid Phe, Trp, Hyp, Hsr,Car, Thr, Arg, Phe, Trp, ATP-ase enzyme Ort, Cav, Asn, Gln, Can, Gln,Asn, Cys and system at the secretory Tau, Djk, GABA, Cys, Ser, Hyp, Sarsurface of the gastric Dcy, Thr, and Sar parietal cell, treatment ofgastric hyperacidity Megestrol 2-100 mg 4-80 mg 20-60 mg Lys, Leu, Ile,Gly, Asp, Lys, Leu, Ile, Gly, Gly, Lys, Sar, Pro, b, c, h, i, j,treatment of anorexia; Preferred Forms 2-100 mg/ 4-80 mg/5 ml 20-60 mg/Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Ser & Ala k, l, n, o, pimproving appetite in Oral Tab/Cap 5 ml 5 ml His, Tyr, Ser, Nor, Arg,Val, Pro, His, Tyr, anorexic and patients Oral Liquid Phe, Trp, Hyp,Hsr, Car, Thr, Arg, Phe, Trp, suffering from AIDS Ort, Cav, Asn, Gln,Can, Gln, Asn, Cys and Tau, Djk, GABA, Cys, Ser, Hyp, Sar Dcy, Thr, andSar Metformin 0.2-3 gm 0.25-1.5 gm 0.5-1 gm Lys, Leu, Ile, Gly, Asp,Lys, Leu, Ile, Gly, Asp, Glu, Lys & Azo o, p treatment of PreferredForms 0.2-1 gm/ 0.25-1.5 mg/ 0.5-1 gm/ Glu, Met, Ala, Val, Pro, Asp,Glu, Met, Ala, dimer hyperglycemia; aids Oral Tab/Cap 5 ml 5 ml 5 mlHis, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, insulin to improve OralLiquid Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp, transport of glucoseOrt, Cav, Asn, Gln, Can, Gln, Asn, Cys and into cells Tau, Djk, GABA,Cys, Ser, Hyp, Sar Dcy, Thr, and Sar Tazarotene 0.01-0.3% 0.02-0.25%0.025-0.125% Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Ser, Hyp, Thr,Lys, & B, c, h, I, j, treatment of psoriasis Preferred Forms Glu, Met,Ala, Val, Pro, Asp, Glu, Met, Ala, Gly k, l, o, p and acne especiallyTopical Gel His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, those caused byPhe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp, pathogenic Ort, Cav, Asn,Gln, Can, Gln, Asn, Cys and microorganisms, allergy Tau, Djk, GABA, Cys,Ser, Hyp, Sar and inflammation Dcy, Thr, and Sar Sumatriptan 5-250 mg10-200 mg 20-125 mg Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Lys,Gly, Ala, Pro, b, c, d, g, h, 5 HT subtype receptor Preferred Forms5-250 mg/ 10-200 mg/ 20-125 mg/ Glu, Met, Ala, Val, Pro, Asp, Glu, Met,Ala, Sar & Val i, j, k, l, n, agonist, treatment of Oral Tab/Cap 5 ml 5ml 5 ml His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, o, p, q migraineheadaches Oral Liquid 1-36 mg/ml 2-24 mg/ml 4-20 mg/ml Phe, Trp, Hyp,Hsr, Car, Thr, Arg, Phe, Trp, IM Injections Ort, Cav, Asn, Gln, Can,Gln, Asn, Cys and Tau, Djk, GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and SarNaratriptan 0.1-10 mg 0.25-5 mg 0.5-4 mg Lys, Leu, Ile, Gly, Asp, Lys,Leu, Ile, Gly, Lys, Gly, Sar, Val, b, h, i, j, k, 5 HT subtype PreferredForms 0.1-10 mg/ 0.25-5 mg/ 0.5-4 mg/ Glu, Met, Ala, Val, Pro, Asp, Glu,Met, Ala, Ala, & Pro l, o, p receptor agonist; Oral Tab/Cap 5 ml 5 ml 5ml His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, treatment of Oral LiquidPhe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp, migraine headaches Ort,Cav, Asn, Gln, Can, Gln, Asn, Cys and Tau, Djk, GABA, Cys, Ser, Hyp, SarDcy, Thr, and Sar Zolmitriptan 0.1-12 mg 0.5-10 mg 1-7.5 mg Lys, Leu,Ile, Gly, Asp, Lys, Leu, Ile, Gly, Lys, Gly, Sar, Val, b, h, i, j, k, 5HT subtype Preferred Forms 1-12 mg/ 0.5-10 mg/ 1-7.5 mg/ Glu, Met, Ala,Val, Pro, Asp, Glu, Met, Ala, Ala, & Pro l, o, p receptor Oral Tab/Cap 5ml 5 ml 5 ml His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, agonist;treatment of Oral Liquid Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp,migraine headaches Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and Tau, Djk,GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and Sar Aspirin 10-1000 mg 20-800 mg25-600 mg Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Ser, Hyp, Thr,Lys, a, b, c, e, f, antipyretic, anti- Preferred Forms 10-1000 mg/ml20-800 mg/ml 25-600 mg/ml Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala,Gly, & Ala g, h, j, k, l, inflammatory, Oral Tab/Cap His, Tyr, Ser, Nor,Arg, Val, Pro, His, Tyr, m, n, o, p, q analgesic, Oral Liquid Phe, Trp,Hyp, Hsr, Car, Thr, Arg, Phe, Trp, thrombolytic; Ort, Cav, Asn, Gln,Can, Gln, Asn, Cys and treatment of Tau, Djk, GABA, Cys, Ser, Hyp, Sarhyperthermia, Dcy, Thr, and Sar myocardial infarction and thrombolysisOlmesartan 1-100 mg 2-80 mg 4-50 mg Lys, Leu, Ile, Gly, Asp, Lys, Leu,Ile, Gly, Ser, Hyp, Thr, Lys, b, h, i, j, k, l, ACE inhibitor, PreferredForms 1-100 mg/ 2-80 mg/ 4-50 mg/5 ml Glu, Met, Ala, Val, Pro, Asp, Glu,Met, Ala, Gly, & Ala o, p treatment of Oral Tab/Cap 5 ml 5 ml His, Tyr,Ser, Nor, Arg, Val, Pro, His, Tyr, hypertension Oral Liquid Phe, Trp,Hyp, Hsr, Car, Thr, Arg, Phe, Trp, Ort, Cav, Asn, Gln, Can, Gln, Asn,Cys and Tau, Djk, GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and Sar Sirolimus0.1-20 mg 0.5-10 mg 1-8 mg Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly,Ser, Hyp, Thr, Lys, b, h, i, j, k, l, immunosuppressant Preferred Forms0.1-20 mg/ 0.5-10 mg/ 1-8 mg/5 ml Glu, Met, Ala, Val, Pro, Asp, Glu,Met, Ala, Gly, & Ala o, p in surgical human Oral Tab/Cap 5 ml 5 ml His,Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, patients with Oral Liquid Phe,Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp, transplants; IM Injections Ort,Cav, Asn, Gln, Can, Gln, Asn, Cys and antibiotic; treating Tau, Djk,GABA, Cys, Ser, Hyp, Sar vitiligo psoriass, Dcy, Thr, and Sar acneTacrolimus 0.1-20 mg 0.2-15 mg 0.25-10 mg Lys, Leu, Ile, Gly, Asp, Lys,Leu, Ile, Gly, Lys, Gly, Ala, Thr, b, c, g, h, i, immunosuppressantPreferred Forms above/5 ml above/5 ml above/5 ml Glu, Met, Ala, Val,Pro, Asp, Glu, Met, Ala, Sar & Pro j, k, l, o, p in surgical human OralTab/Cap 1-20 mg/ml 2-15 mg/ml 2.5-8 mg/ml His, Tyr, Ser, Nor, Arg, Val,Pro, His, Tyr, patients with Oral Liquid Phe, Trp, Hyp, Hsr, Car, Thr,Arg, Phe, Trp, transplants; IV Infusions Ort, Cav, Asn, Gln, Can, Gln,Asn, Cys and antibiotic; treating Tau, Djk, GABA, Cys, Ser, Hyp, Sarvitiligo psoriass, Dcy, Thr, and Sar acne Pimecrolimus 0.1-20 mg 0.2-15mg 0.25-10 mg Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Lys, Gly,Ala, Thr, b, c, g, h, i, immunosuppressant Preferred Forms above/5 mlabove/5 ml above/5 ml Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Sar &Pro j, k, l, o, p in surgical human Oral Tab/Cap 0.01-10% 0.1-5% 0.5-2%His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, patients with Oral LiquidPhe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp, transplants; Ointment CreamOrt, Cav, Asn, Gln, Can, Gln, Asn, Cys and antibiotic treating Tau, Djk,GABA, Cys, Ser, Hyp, Sar vitiligo psoriass, Dcy, Thr, and Sar acneClopidogrel 10-250 mg 20-125 mg 25-100 mg 20-125 mg 25-100 mg Ser, Hyp,Thr, Lys, b, c, h, i, j, treatment of Preferred Forms 10-250 mg/ 20-125mg/ 25-100 mg/ 20-125 mg/5 ml 25-100 mg/5 ml Ala, & Gly k, l, m, o, p, qmyocardial Oral Tab/Cap 5 ml 5 ml 5 ml infections Oral LiquidAmphotericin B 0.5-20 mg/kg/ 1-15 mg/kg/ 2-10 mg/kg/ Lys, Leu, Ile, Gly,Asp, Lys, Leu, Ile, Gly, Ser, Hyp, Thr, Lys, b, c, g, i, j, treatment offungus, Preferred Forms day day day Glu, Met, Ala, Val, Pro, Asp, Glu,Met, Ala, Ala, & Gly l, m, n, o, p, q expecially those IV Infusion0.01-10% 0.1-5% 0.5-2% His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr,acting on cell Topical Cream Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe,Trp, membrane changing Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and itspermeability Tau, Djk, GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and SarTenofovir 10-900 mg 50-750 mg 100-500 mg Lys, Leu, Ile, Gly, Asp, Lys,Leu, Ile, Gly, Lys, Gly, Ala, Pro, b, c, h, i, j, inhibitor of HIVPreferred Forms 10-900 mg/ 50-750 mg/ 100-500 mg/ Glu, Met, Ala, Val,Pro, Asp, Glu, Met, Ala, Ser & Sar k, l, o, p virus, treatment of OralTab/Cap 5 ml 5 ml 5 ml His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, AIDSinfections Oral Liquid Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp, Ort,Cav, Asn, Gln, Can, Gln, Asn, Cys and Tau, Djk, GABA, Cys, Ser, Hyp, SarDcy, Thr, and Sar Unoprostone 0.01-1% 0.05-0.5% 0.01-0.25% Lys, Leu,Ile, Gly, Asp, Lys, Leu, Ile, Gly, Ser, Hyp, Thr, Tyr, b, c, h, i, j,treatment of Preferred Forms Glu, Met, Ala, Val, Pro, Asp, Glu, Met,Ala, Pro, & Lys k, l, n, o, p, q glaucoma, Ocular Drops His, Tyr, Ser,Nor, Arg, Val, Pro, His, Tyr, especially caused by Phe, Trp, Hyp, Hsr,Car, Thr, Arg, Phe, Trp, age; lowers Ort, Cav, Asn, Gln, Can, Gln, Asn,Cys and intraocular pressure Tau, Djk, GABA, Cys, Ser, Hyp, Sar Dcy,Thr, and Sar Fulvestrant 2-1250 mg/ 10-1000 mg/ 20-500 mg/ Lys, Leu,Ile, Gly, Asp, Lys, Leu, Ile, Gly, Gly, Lys, Pro, Ala, B, c, g, j, l,treating cancer, Preferred Forms 5 ml 5 ml 5 ml Glu, Met, Ala, Val, Pro,Asp, Glu, Met, Ala, Val & Sar o, p especially breast IM Injection His,Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, cancer Phe, Trp, Hyp, Hsr, Car,Thr, Arg, Phe, Trp, Ort, Cav, Asn, Gln, Can, Gln, Asn, Cys and Tau, Djk,GABA, Cys, Ser, Hyp, Sar Dcy, Thr, and Sar Cefditoren 20-500 mg 100-400mg 150-300 mg Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Ser, Hyp,Thr, Gly, b, c, h, i, j, antibiotics, Preferred Forms 20-500 mg/ 100-400mg/ 150-300 mg/ Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Lys & Alak, l, o, p especially inhibits Oral Tab/Cap 5 ml 5 ml 5 ml His, Tyr,Ser, Nor, Arg, Val, Pro, His, Tyr, H. influenzae; Oral Liquid Phe, Trp,Hyp, Hsr, Car, Thr, Arg, Phe, Trp, Haemophilus para- Ort, Cav, Asn, Gln,Can, Gln, Asn, Cys and influenzae, Tau, Djk, GABA, Cys, Ser, Hyp, Sarstreptococcus, Dcy, Thr, and Sar Maraxella catarrhalis; treatment ofbronchitis, pharyngitis, tonsillitis, skin infections Efavirenz 0.2-1.2gm 300-800 mg 400-750 mg Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly,Gly, Lys, Pro, Ala, b, c, h, i, j, inhibitor of HIV-1 Preferred Forms0.2-1.2 gm/ 300-800 mg/ 400-750 mg/ Glu, Met, Ala, Val, Pro, Asp, Glu,Met, Ala, Sar, & Val k, l, o, p specific, non- Oral Tab/Cap 5 ml 5 ml 5ml His, Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, nucleoside, reverse OralLiquid Phe, Trp, Hyp, Hsr, Car, Thr, Arg, Phe, Trp, transcirptase; Ort,Cav, Asn, Gln, Can, Gln, Asn, Cys and treatment of AIDS Tau, Djk, GABA,Cys, Ser, Hyp, Sar infections Dcy, Thr, and Sar Eplerenone 10-250 mg15-200 mg 20-150 mg Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Ser,Hyp, Thr, Lys, b, c, h, i, j, treatment of Preferred Forms 10-250 mg/15-200 mg/ 20-150 mg/ Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Gly &Val k, l, o, p hypertension, blocks Oral Tab/Cap 5 ml 5 ml 5 ml His,Tyr, Ser, Nor, Arg, Val, Pro, His, Tyr, binding of Oral Liquid Phe, Trp,Hyp, Hsr, Car, Thr, Arg, Phe, Trp, aldosterone to Ort, Cav, Asn, Gln,Can, Gln, Asn, Cys and mineralo-corticoid Tau, Djk, GABA, Cys, Ser, Hyp,Sar receptors Dcy, Thr, and Sar Treprostinil 0.1-100 mg/ml 0.2-50 mg/ml0.5-20 mg/ml Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Gly, Lys, Val,Hyp, b, c, g, h, i, Inhibits platelet Preferred Forms 10-1000 mg 20-800mg 25-500 mg Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Thr & Ser j,k, l, o, p aggregation and SC infusion His, Tyr, Ser, Nor, Arg, Val,Pro, His, Tyr, vasodilation of Oral Tab/Cap Phe, Trp, Hyp, Hsr, Car,Thr, Arg, Phe, Trp, systemic and Ort, Cav, Asn, Gln, Can, Gln, Asn, Cysand pulmonary vascular Tau, Djk, GABA, Cys, Ser, Hyp, Sar bed, treatmentof Dcy, Thr, and Sar cardiovascular related conditions Adefovir 1-100 mg2-50 mg 5-20 mg Lys, Leu, Ile, Gly, Asp, Lys, Leu, Ile, Gly, Lys, Gly,Val, Ser, b, c, h, i, j, HIV reverse Preferred Forms 1-100 mg/ 2-50 mg/5-20 mg/ Glu, Met, Ala, Val, Pro, Asp, Glu, Met, Ala, Hyp, & Pro k, l,o, p transcriptase Oral Tab/Cap 5 ml 5 ml 5 ml His, Tyr, Ser, Nor, Arg,Val, Pro, His, Tyr, inhibitors; treatment Oral Liquid Phe, Trp, Hyp,Hsr, Car, Thr, Arg, Phe, Trp, of HIV infections Ort, Cav, Asn, Gln, Can,Gln, Asn, Cys and and AIDS Tau, Djk, GABA, Cys, Ser, Hyp, Sar Dcy, Thr,and SarAbove table is not exhaustive, and not final. Inventor has givenexamples of certain drugs and proposed compostions and dosages, androute of administration. However, above table is typical and does notconstraint the scope of this invention.

As used herein, the term “amino acid derivative” or “drug derivative”and like terms refer to the drug bonded to an amino acid residue. Inaddition, as used herein, the term L-amino acid refers to an amino acidwherein the asymmetric carbon atom attached to the amino and the carboxygroup is in the L-configuration. For purpose of this specification, italso includes glycine.

As indicated hereinabove, the amino acid derivatives of the presentinvention have the same utility as the underlying drug which is notbonded to the amino acid moiety. The amino acid derivative of thepresent invention possesses at least one enhanced quality identifiedhereinabove relative to the same drug without the amino acid linkage.

Without wishing to be bound it is believe that the amino acidderivatives of the present invention is the active agent in vivo.Alternatively, the amino acid moiety may be cleaved off in vivo, therebymaking the active agent the drug itself in vivo. Alternatively, acombination of both may occur in vivo. However, whichever happens invivo, when the amino acid derivative of the present invention isadministered to a patient, at least one of the improved qualitiesenumerated hereinabove relative to the drug without the amino acidlinkage is realized.

The following non-limiting examples further illustrate the invention:

Synthesis of Various Amino Acid Derivatives of Selected Drugs 1.Propofol Derivatives

Propofol (2,6-diisopropylphenol) is a low molecular weight phenol whichwidely used as a central nervous system anesthetic, and posses sedativeand hypnotic activities. It is administered intravenously in theinduction and maintenance of anesthesia and/or sedation in mammals. Themajor advantages of Propofol are that it can induce anesthesia rapidly,minimal side effects and upon withdrawal, the patient recovers quicklywithout prolonged sedation.

Propofol has been shown to have a large number of therapeuticapplications, which are quite varying and somewhat surprising. Forexample, it has been shown to be an effective antioxidant, anti-emetic,anti-pruritic, anti-epileptic, anti-inflammatory, and even seems topossess anti-cancer properties.

Mechanism of Action:

The mechanism of action of Propofol has been extensively studied. Itscentral nervous system anesthetic activity has been shown to be relatedits high affinity for a specific subclass of GABA receptors (Collins G.G. S., 1988, Br. J. Pharmacology. 542, 225-232). However, there are anumber of different receptors in the brain which are substrates forpropofol, hence its varied activities.

Propofol also has significant biological effect as an antioxidant.Because of this generalized activity of propofol, it is theoreticallyuseful in the treatment of a number of inflammatory processes whereoxidation is an important factor. For example, cyclooxygenase mediatedprostaglandin synthesis results in inflammation. By inhibiting oxidationin the respiratory tract, one could use propofol in the treatment ofacid aspiration, adult/infant respiratory distress syndrome, airwayobstructive diseases, asthma, cancer and a number of other similarpathological conditions.

Since oxidative tissue damage is a very common occurrence, it has beensuggested that propofol is useful in the treatment of Parkinson'sdisease, Alzheimer disease, Friedrich's disease, Huntington's disease,multiple sclerosis, amyotrophic lateral sclerosis, spinal chordinjuries, and various other neurodegenerative diseases.

Propofol is currently available in the US market as an intravenousemulsion marketed by Astra Zenaca under the brand name Diprivan®. It isone the most widely used short acting central nervous system anestheticsin the market. The concentration of propofol is 10 mg/mL innon-pyrogenic sterile emulsion and the formula contains soybean oil,glycerol, egg lecithin, disodium edetate and sodium hydroxide.

A significant disadvantage of Propofol is that it is completelyinsoluble in water. Even at very low concentrations of 10 mg/mL, thedrug precipitates out of an aqueous solution in room temperature.Therefore, manufacturers of this formulation use heroic methods toemulsify this product in water using extraordinarily complex and toxicemulsifying agents. For example, manufacturers of the IV formulationsuse egg lecithin, Cremaphor L®, castor oil, and other similaremulsifiers.

However, use of such emulsifiers is associated with number of problems.It is well known that various types of Cremaphor L® emulsifiers canprecipitate allergic reactions. Egg lecithin and castor oil have beenshown to produce anaphylactic shock in some patients. Furthermore,maintenance of stability of propofol in these emulsions is short livedand more expensive. Moreover, the presence of egg lecithin and castoroil make the emulsion prone to microbial growth. It may be possible todissolve propofol in water by complexing it with cyclodextrin, butcyclodextrin has not been approved by the FDA for use in intravenoustherapy.

Heretofore, no one has made a safe derivative of propofol. The Britishpatents 1,102,011, and 1,160,468 and U.S. Pat. No. 3,389,138 describethe various phenol esters of amino acids, wherein the propofol isattached to a number of side chains which when released in the bodyproduce toxic effects.

U.S. Pat. No. 6,451,854 describe a number of substituted alpha aminoacetic acid esters of propofol, wherein propofol and the side chain weresubstituted with a number of different chemical groups. All theN,N-disubstituted glycine esters of propofol have not shown to benon-toxic and many of the compounds described are derivative ofpropofol. Thus when released in the body after the cleavage of ester bythe enzymes, many of the active drugs released are not propofol, andhence they do not possesses any toxicity data and are entirely newmolecules with unknown therapeutic efficacy in man.

In a published paper on the water soluble salts of amino acid esters ofthe anesthetic agent propofol, (Int. J. Pharmaceutics, 175[2]: 195-204,1998) authors have synthesized a number of water soluble derivatives ofpropofol. However, when these derivatives are cleaved by esteraseenzymes, substituted non-natural amino acids with unknown toxicityprofile are released in the body.

Until now there has been no pharmaceutical preparation available in themarket that can deliver propofol without harmful side effects. Thepresent inventor has produced a number of water soluble, non-toxicderivatives of propofol which are suitable for delivering propofol inthe body without any harmful side effects and without the needs fortoxic and expensive additives, solubilizers and emulsifiers.Accordingly, in one aspect, the present invention is directed to a classof derivatives of Propofol. The derivative consists of the carboxylgroup of an amino acid esterified to the free hydroxyl group present onthe propofol molecules.

More specifically, one aspect of the present invention is directed to,the compounds of the formula:

or pharmaceutically acceptable salts thereof; wherein AA is an aminoacid, in which the carboxyl group of AA is reacted with the hydroxylgroup of the Propofol.

In another aspect, the present invention is also directed to apharmaceutical composition comprising a therapeutically effective amountof the various Propofol amino acid derivatives especially Proline Esterdrugs and a pharmaceutical carrier therefor. Preferred amino acidderivatives are indicated in Table I.

In another embodiment, the present invention is directed to a method oftreating a patient in need of propofol therapy, which method comprisesadministering to said patient an effective amount of the Propofol.

In a further embodiment, the present invention is directed to a methodof enhancing the solubility of propofol in an aqueous solutioncomprising reacting the hydroxyl functionality of the Propofol with aamino acid, especially a naturally occurring amino acid, and isolatingthe product thereof.

In a still further embodiment, the present invention is directed to amethod of substantially and in a therapeutically efficacious manner,reducing or eliminating the potential toxic side effects of currentformulations containing toxic excipients when administered to a patientwhich comprises reacting the hydroxyl functionality of the propofolmolecule with carboxyl function of the amino acid, especially thepreferred and most preferred amino acids depicted in Table I, to form anester covalent bond respectively and isolating the product thereof andadministering said product to the patient.

Moreover, the inventor has discovered that when unsubstituted naturallyoccurring amino acids are esterified to propofol, the resultingderivatives are highly water soluble, (>200 mg/L in water), releasenon-toxic amino acids upon cleavage in the body and require none of thetoxic emulsifier, additives and other excipients.

Furthermore, the inventor has found that the amino acid derivatives ofpropofol of the present invention are highly effective central nervoussystem anesthetics. Thus the current amino acid derivatives areeffective central nervous system anesthetics, with or without releasingthe active parent drug.

The amino acid esters of the present invention are at least 10 timesmore soluble that propofol in water in room temperature. Especially theglycine, proline and lysine esters of propofol are soluble at the rangeof more than 100 mg/ml, and in case of lysine it is greater than 250mg/mL.

The amino acid derivatives of the present invention are not expected topossess any antioxidant activity due to blockage of the phenolic groupresponsible for such; however the present inventor has found that theamino acid derivatives of propofol are effective anesthetics with orwithout releasing propofol. Further, the Propofol Proline Ester drugsdescribed, when administered in vivo, maintains its pharmacological andanti-oxidant properties.

The amino acid derivative of propofol of the present invention clearlyprovides a number of advantages over propofol, for example, all of theside chains cleaved from these derivatives are naturally occurringessential amino acids and hence are non-toxic. This results in hightherapeutic index. Secondly the derivatives are readily cleaved in thebody to release propofol, however, if not cleaved the amino acidderivatives exhibit the same utility. Furthermore, due to their highwater solubility, they can be easily administered by either forming anin-situ solution just before IV administration using lyophilized sterilepowder or providing the drug in solution in prefilled syringe or bottlesfor infusion. The amino acid esters are more stable than propofol sinceOH group in propofol is blocked thereby prevents oxidation. For example,the Propofol Proline Ester drugs of the present invention are moreeffective then propofol itself without the toxicity and otherpharmaceutical problem associated with current marketed formulations.

The amino acid derivatives of propofol of the present invention possessanti-inflammatory, anti-oxidant, anti-cancer, anti-convulsive,anti-emetic and anti-pruritic properties.

These derivatives of propofol of the present invention are effective intreating diseases or conditions in which Propofol normally are used. Thederivatives disclosed herein may or may not be transformed within thebody to release the active compound, and can enhance the therapeuticbenefits of the Propofol moiety by reducing or eliminatingbiopharmaceutical and pharmacokenetic barriers associated with each ofthem. However it should be noted that these derivatives themselves willhave sufficient activity without releasing any active drug in themammals. Since the derivatives are more soluble in water then Propofol,it does not need to be associated with a carrier vehicle, such asalcohol or castor oil which may be toxic or produce unwanted sidereactions. Moreover, oral formulations containing the derivatives ofPropofol are absorbed into the blood and are quite effective.

Thus, the amino acid derivative of the present invention, e.g., theamino acid derivative of propofol, enhances the therapeutic benefits byremoving biopharmaceutical and pharmacokenetic barriers of existingdrugs.

Furthermore, the amino acid derivatives, e.g., the amino acidderivatives of propofol, are easily synthesized in high yields usingreagents which are readily and commercially available.

Overview:

The procedure for the synthesis of the glycine, L-proline, and L-lysineesters of Propofol is depicted hereinbelow. However, these are exemplaryand any amino acid derivative thereof can be prepared using thefollowing methodology. The complete procedure and analytical data isgiven in the Experimental Section. In general, as shown in the followingscheme Propofol (10 g) was coupled with the N-Boc protected amino acid(1 equivalent) in the presence of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC) inthe presence of a catalytic amount of 4-(N,N-dimethyamino)-pyridine(DMAP). The EDC was removed by extraction with water. After drying oversodium sulfate, filtration, and concentration thereof, the crudeprotected amino acid ester of Propofol was purified by flashchromatography to generate the protected esters in 50-60% yield. Theprotecting groups were then removed by stirring the protected esters indiethyl ether saturated with hydrochloric acid (gas) at roomtemperature. Yields for the deprotection step were generally 60-95%.After filtration and drying the hydrochloride salts of the glycine andL-proline esters of Propofol did not require additional purification.The hydrochloride salt of the L-lysine-Propofol ester was crystallizedonce from ethanol to remove a trace of mono-protected L-lysine-Propofolester.

Synthetic Sequence:

Synthesis of the Glycine, L-Proline, and L-Lysine Esters of Propofol a)EDC, DMAP, CH₂Cl₂; b) HCl (g), Et₂O Experimental Section

The synthesis of SPI0010, SPI0011 and SPI0013 were conducted in batches.Generally a small-scale experiment was performed first followed by alarger batch. Reagents mentioned in the experimental section werepurchased at the highest obtainable purity from Aldrich, Acros, orBachem, except for solvents, which were purchased from either FisherScientific or Mallinkrodt.

1) SPI0010

Propofol (9.98 g, 55.97 mmole) was dissolved in dichloromethane (200 mL)at room temperature, under an argon atmosphere.N-t-Butyloxocarbonyl-glycine (11.2 g, 63.91 mmole) was added along with1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC, 11.1g, 57.9 mmole) and 4-(N,N-dimethylamino)-pyridine (DMAP, 1.5 g, 12.27mmole). After stirring for 21 hours under an argon atmosphere at roomtemperature, water (200 mL) was added and the layers were separated. Thedichloromethane layer was washed with water (200 mL) and dried for 1hour over sodium sulfate (5 g). After filtration and concentration underreduced pressure, the remaining oil was purified by flash chromatographyon silica gel (250 g), eluting with hexanes/ethyl acetate (10:1). Theprocedure generated the protected N—BOC protected glycine ester ofPropofol as a white solid (11.34 g, 60% yield).

tert-Butoxycarbonylamino-acetic acid 2,6-diisopropylphenyl ester:

¹H NMR (300 MHz, CDCl₃): δ=7.25-7.13 (m, 3H), 5.18 (br s, 1H), 4.22 (d,2H, J=5.7 Hz), 2.89 (m, 2H), 1.46 (s, 9H), 1.18 (d, 12H, J=6.9 Hz).

¹³C NMR (75 MHz, CDCl₃): δ=169.35, 155.75, 145.22, 140.35, 126.90,124.14, 80.32, 42.66, 28.54, 27.79, 23.57.

The Propofol-Boc-gycine ester (11.28 g, 33.6 mmole) was dissolved inanhydrous diethyl ether (200 mL) at room temperature. Hydrochloric acid(gas) was passed through the solution for 45 minutes while stirring. Themixture was allowed to stir at room temperature for 48 hours under anargon atmosphere. After 48 hours hexanes (200 mL) were added and theprecipitate was filtered. The white solid was dried under high vacuumfor 5 hours at 88° C. The experiment produced SPI0010 (8.73 g, 95%yield, purity 99.9% by HPLC) as a white solid.

Amino-acetic acid 2,6-diisopropyl-phenyl ester, hydrochloride

¹H NMR (300 MHz, CDCl₃): δ=8.77 (br s, 3H), 7.20-7.08 (m, 3H), 4.14 (m,2H), 2.87 (m, 2H), 1.11 (d, 12H, J=7 Hz).

¹³C NMR (75 MHz, CDCl₃): δ=166.42, 144.84, 140.42, 127.10, 124.06,40.47, 27.61, 23.55.

CHN Analysis:

calc.: C, 61.87; H, 8.16; N, 5.15. found: C, 61.14; H, 8.20; N, 5.14.

2) SPI0011

Propofol (10.03 g, 56.23 mmole) was dissolved in dichloromethane (100mL) at room temperature, under an argon atmosphere.N-t-Butyloxocarbonyl-L-proline (14.04 g, 65.22 mmole) was added alongwith 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC,11.95 g, 62.33 mmole) and 4-(N,N-dimethylamino)-pyridine (DMAP, 1.1 g,9.0 mmole). After stirring for 3 hours under an argon atmosphere at roomtemperature, water (100 mL) was added and the layers were separated. Thedichloromethane layer was washed again with water (100 mL) and dried for1 hour over sodium sulfate (5 g). After filtration and concentrationunder reduced pressure, the remaining oil was purified by flashchromatography on silica gel (250 g), eluting with hexanes/ethyl acetate(10:1). The procedure generated the protected N-BOC protected L-prolineester of Propofol as a clear oil (11.34 g, 66% yield) that solidified onstanding in the freezer.

Pyrrolidine-1,2-dicarboxylic acid 1-tert-butyl ester2-(2,6-diisopropyl-phenyl)ester

¹H NMR (300 MHz, CDCl₃): δ=7.31-7.20 (m, 3H), 4.73 (m, 1H), 3.70-3.50(m, 2H), 3.20-2.94 (m, 2H), 2.46-2.20 (m, 2H), 2.20-2.0 (m, 2H), 1.55(m, 9H), 1.25 (m, 12H).

¹³C NMR (75 MHz, CDCl₃): δ=171.87, 171.01, 154.34, 153.93, 145.35145.23, 140.06, 140.21, 126.69, 126.53, 123.95, 80.28, 79.89, 59.14,46.67, 46.42, 31.10, 30.17, 28.61, 28.56, 28.56, 27.44, 27.18, 23.47.

The Propofol-Boc-L-proline ester (13.95 g, 37.14 mmole) was dissolved inanhydrous diethyl ether (100 mL) at room temperature. Hydrochloric acid(gas) was passed through the solution for 60 minutes while stirring. Themixture was allowed to stir at room temperature for 22 hours under anargon atmosphere. After 22 hours, hexane (50 mL) was added and theprecipitate was filtered. The white solid was dried under high vacuumfor 5 hours at 88° C. The experiment produced SPI0011 (9.1 g, 81% yield,purity 99.1% by HPLC) as a white solid.

Pyrrolidine-2(S)-carboxylic acid 2,6-diisopropyl-phenyl ester,hydrochloride

¹H NMR (300 MHz, CDCl₃): δ=10.15 (br s, 2H), 7.27-7.14 (m, 3H), 4.78 (t,1H, J=7.8 Hz), 3.56 (m, 2.85 (m, 2H), 2.64 (m, 1H), 2.40 (m, 1H), 2.20(m, 1H), 2.05 (m, 1H), 1.18 (m, 12H).

¹³C NMR (75 MHz, CDCl₃): δ=168.30, 144.23, 139.74, 126.98, 123.96,51.58, 38.21, 29.32, 26.64, 26.18, 23.71, 23.02, 21.67.

CHN Analysis:

calc.: C, 65.48; H, 8.40; N, 4.49. found: C, 65.50; H, 8.43; N, 4.50.

3) SPI0013

The dicyclohexylamine salt of di-N-boc-L-lysine (23.62 g, 0.0447 mole)was added to diethyl ether (200 mL) and potassium hydrogen sulfate (9.14g) in water (200 mL) that was cooled in an ice/water bath. Afterstirring for 20 minutes, the layers were separated. The ether layer wasextracted three times with cold water (100 mL). The ether layer was thendried over sodium sulfate (15 g) for one hour, filtered, andconcentrated under reduced pressure. The procedure generated the freeacid of N,N′-di-boc-L-lysine (15.5 g, 100% recovery).

Propofol (8.0 g, 45 mmole) was dissolved in dichloromethane (100 mL) atroom temperature, under an argon atmosphere.N,N′-di-t-Butyloxocarbonyl-L-lysine (15.5 g, 44.7 mmole) was added alongwith 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC,8.62 g, 45 mmole) and 4-(N,N-dimethylamino)-pyridine (DMAP, 0.55 g, 4.5mmole). After stirring for 3 hours under an argon atmosphere at roomtemperature, water (100 mL) was added and the layers were separated. Thedichloromethane layer was washed again with water (100 mL) and dried for1 hour over sodium sulfate (5 g). After filtration and concentrationunder reduced pressure, the remaining oil was purified by flashchromatography on silica gel (250 g), eluting with hexanes/ethyl acetate(9:1). The procedure generated the protected N—BOC protected L-lysineester of Propofol as a white foam (12.42 g, 55% yield).

2(S),6-Bis-t-butoxycarbonylamino-hexanoic acid 2,6-diisopropyl-phenylester

¹H NMR (300 MHz, CDCl₃): δ=7.28-7.15 (m, 3H), 5.22 (d, 1H, J=8.4 Hz),4.70 (m, 1H), 4.59 (m, 1H), 3.17 (m, 2H), 2.93 (m, 2H), 2.09 (m, 1H),1.86 (m, 1H), 1.67-1.54 (m, 4H), 1.48 (s, 9H), 1.46 (s, 9H), 1.20 (m,12H).

¹³C NMR (75 MHz, CDCl₃): δ=171.82, 156.10, 155.65, 145.25, 140.30,126.80, 124.03, 80.14, 79.28, 53.76, 40.29, 32.09, 28.66, 28.54, 27.48,23.91, 23.10.

The Propofol-di-Boc-L-lysine ester (12.34 g, 24.37 mmole) was dissolvedin anhydrous diethyl ether (250 mL) at room temperature. Hydrochloricacid (gas) was passed through the solution for 60 minutes while stirringand cooling in an ice/water bath. The mixture was allowed to stir atroom temperature for 48 hours under an argon atmosphere. After 48 hoursthe precipitate was filtered and crystallized from ethanol (100 mL). Thewhite solid was dried under high vacuum for 4 hours at 90° C. Theexperiment produced SPI0013 (5.5 g, 60% yield, purity 98.6% by HPLC) asa white solid.

2(S),6-Diamino-hexanoic acid 2,6-diisopropyl-phenyl ester,dihydrochloride

¹H NMR (300 MHz, CDCl₃): δ=9.05 (br s, 3H), 8.35 (br s, 3H), 7.26-7.13(m, 3H), 4.43 (t, 1H, J=6 Hz), 3.0-2.6 (m, 4H), 2.09 (m, 2H), 1.80-1.50(m, 4H), 1.10 (d, 12H, J=7 Hz).

¹³C NMR (75 MHz, CDCl₃): δ=168.30, 144.23, 139.74, 126.98, 123.96,51.58, 38.21, 29.32, 26.64, 23.71, 23.02, 21.67.

CHN Analysis:

calc.: C, 56.99; H, 8.50; N, 7.38. found: C, 56.48; H, 8.56; N, 7.30.

Anasthetic Effect in Male Albino Mice for derivatives of Propofol

The present study was conducted to evaluate the efficacy of newformulations of propofol injection using righting reflex as an index inalbino mouse. Propofol injection served as reference control. Malealbino mice were exposed to propofol injection and to 3 new formulationsof propofol at 3 dose levels—low, intermediate and high dose levels.However the doses of low, intermediate and high for differentformulations and reference control were different.

The different doses for each formulation and reference control wereselected based on the recommendation of Sponsor. All the doses wereexpressed as propofol molar equivalents. The doses used for the mainexperiment for different formulations and reference control werepresented below.

TABLE 2 Low Dose Intermediate High Dose Test Item (mg/kg) Dose (mg/kg)(mg/kg) Propofol Lysine Ester 40.0 45.0 50.0 Propofol Glycine Ester 30.035.0 40.0 Propofol Proline Ester 25.0 30.0 35.0 Propofol Injection 10.015.0 20.0 (Reference control)

Efficacy (Anesthetic Property)

The efficacy in terms of time required to regain the righting reflex atdifferent dose levels—low, intermediate and high dose for differentformulations and propofol injection are presented below.

TABLE 3 Low Dose Intermediate Dose High Dose Formulation 1 0.00 ± 0.000.00 ± 0.00 546.00 ± 0.00  Formulation 2 532.10 ± 217.61 299.80 ± 211.32641.00 ± 137.86 Formulation 3 282.00 ± 162.09 421.70 ± 112.92 237.25 ±197.47 Propofol injection 20.20 ± 43.42 123.40 ± 33.70  177.70 ± 70.31 (Reference control)

All doses produced statistically significant results at p<0.05.

Low Dose

Propofol Glycine Esterl was found to be more effective followed byformulation 3 and reference control. Formulation 1 did not show anyanesthetic effect.

No mortality was observed at low dose level with different formulationsor propofol injection.

Intermediate Dose

Propofol Proline Ester ester was found to be more effective followed byPropofol Glycine Esterl and reference control. Propofol lysine ester didnot show any anesthetic effect.

High Dose

Propofol Glycine Esterl was found to be more effective followed bypropofol lysine ester, Propofol Proline Esterraline ester and referencecontrol.

Dose Response Relation Propofol Lysine Ester

The statistical analysis showed a significant improvement at 5%significance level in the relative efficacy for the high dose whencompared to low dose group.

Propofol Glycine Ester

The responses to the dose levels were not statistically evident from theanalysis, though the comparisons showed significance at 5% level for middose to low dose.

Propofol Proline Ester

No significant dose—response effects were evident from the statisticalanalysis.

Propofol Injection (Reference Control)

For reference control group, the dose response effect were statisticallysignificant and clearly evident.

In conclusion, it was observed that based on the time required to regainthe righting reflex, Propofol Glycine Ester and the lysine ester haverelatively better efficacy than the other two derivatives (PropofolProline ester and the drug) in inducing anesthesia in albino mice.

14-day Chronic Toxicity Studies in Albino Mice using Propofol GlycineEster and Proline Ester:

The present study was conducted to evaluate the relative safety of thetwo new formulations of Propofol injection

Propofol Glycine Ester

Propofol Proline Ester

by conducting a 14 day repeated dose toxicity study through intravenousroute using albino mice as test system. Propofol injection served asreference control.

Propofol and its derivatives above were administered to albino mice(Swiss strain), through intravenous route daily for a period of 14 daysusing sterile sodium chloride solution as vehicle. The study wasconducted at one dose level only along with a vehicle control group asper the recommendation of the Sponsor.

The doses used in the present study for the two amino acid ester andPropofol injection were different and presented below. The doses wereselected as per the recommendation of the Sponsor. The test doses areexpressed as Propofol molar equivalents.

TABLE 4 Dose Equivalent [in terms of weight of the Propofol] Test itemTest Item Group (mg/kg) [mg/kg] Vehicle Vehicle Control 0.0 0.0(Propofol Glycine Test Group 1 30.0 45.60 Ester) (Propofol Proline Testgroup 2 25.0 43.75 Ester) Propofol injection Reference Control 15.015.00

The salient features of the study are as follows:

1. All the animals of control and the test group 1 (Propofol GlycineEster), test group 2 (Propofol Proline Ester) and reference controlgroup (Propofol Injection) survived through the dosing period of 14days.2. None of the animals of the vehicle control and the test group 1(Propofol Glycine Ester), test group 2 (Propofol Proline Ester) andReference Control (Propofol Injection) group exhibited any clinicalsymptoms of toxicity. However animals dosed with the amino acid estersof Propofol or reference control showed anesthetic effect immediatelyafter dosing. This effect was attributed to the pharmacological propertyof the amino acid esters or reference control.3. Body weight gain of animals of test group 2 (Propofol Proline Ester)and reference control (Propofol Injection) was found to be normal andcomparable to that of vehicle control group of animals. However, thebody weight gain was reduced in both the sexes of test group 1 (PropofolGlycine Ester). Males and females of test group 1 (Propofol GlycineEster) showed a reduced body weight gain of 25.87% and 26.94%respectively compared to vehicle control group animals.4. Feed intake of the animals of test group 1 (Propofol Glycine Ester),test group 2 (Propofol Proline Ester) and reference control (Propofolinjection) was found to be normal and comparable to that of vehiclecontrol group.5. Results of hematological analysis revealed the following changes inthe animals of different groups when compared to reference control.

Propofol Glycine Ester

The granulocytes counts significantly increased [p<0.05] in femaleanimals of Propofol Glycine Ester when compared to the female animals ofPropofol injection (RC).

The MCV significantly decreased [p<0.05] in female animals of PropofolGlycine Ester when compared to the female animals of Propofol injection(RC).

Propofol Proline Ester

The granulocytes significantly decreased [p<0.05] in male animals ofPropofol Proline Ester when compared to the male animals of Propofolinjection (RC).

The lymphocytes counts significantly increased [p<0.05] in male animalsof Propofol Proline Ester when compared to the male animals of Propofolinjection (RC).

The MCV significantly decreased [p<0.05] in female animals of PropofolProline Ester when compared to the female animals of Propofol injection(RC).

Vehicle Control

The lymphocytes counts significantly increased [p<0.05] in male andfemale animals of vehicle control when compared to the male and femaleanimals of Propofol injection (RC).

6. Results of clinical chemistry analysis revealed the following changesin the animals of different groups when compared to reference control.

Propofol Proline Ester

The level of urea significantly decreased [p<0.05] in female animals ofPropofol Proline Ester when compared to the female animals of Propofolinjection (RC). The level of sodium significantly increased [p<0.05] inmale animals of Propofol Proline Ester when compared to the male animalsof Propofol injection (RC).

Vehicle Control

The cholesterol level significantly decreased [p<0.05] in female animalsof vehicle control when compared to the female animals of Propofolinjection (RC).

Thus the results the present study showed that the

-   -   behavior of animals,    -   body weight gain (except in male and female animals of Propofol        Glycine Ester)    -   feed consumption,    -   Organ weights (absolute and relative)    -   histopathology of different organs        of the animals treated with Propofol Glycine Ester and Propofol        Proline Ester were found to be normal and comparable to that of        the animals of the reference group.

II. Amino Acid Derivatives of Non-Steroidal Anti-Inflammatory Drugs(NSAIDs)

The NSAIDs comprise a class of structurally distinctive carboxylic acidmoiety attached to a planar aromatic functionality. Examples include:acetyl salicyclic acid, salicyclic acid, diflunisal, ibuprofen,fenoprofen, carprofen, flurbiprofen, ketoprofen, naproxen, sulindac,indomethacin, etodolac, tolmetin, ketorolac, diclofenac, andmeclofenamate. The NSADIs posess anti-inflammatory, analgesic,antipyretic and anti-clotting activity.

Examples of the chemical structures of this uniques class of compoundsshowing wide variety of pharmacological activities are shown below.

NSAIDs are widely used for the treatment of acute and chronic pain,management of edema, tissue damage resulting from inflammatory jointdiseases and also, effective anti-clotting agents in the treatment ofmyocardial infraction. A number of the agents also possess antipyreticactivity in addition to analgesic and anti-inflammatory action, thususeful in reducing fever.

Some drugs in the above group have also been prescribed for RheumatoidArthritis, Osteoarthritis, acute gout, ankolysing spondylitis, anddysmenorrhea.

Mechanism of Action:

The major mechanism by which the NSAIDs produce their therapeutic effectis via inhibition of prostaglandin synthesis. Specifically NSAIDsinhibit cyclooxygenases, such as COX-1 and COX-2 enzymes, where thesetwo enzymes are responsible for synthesis of prostaglandins. While COX-1enzyme is important for the regulation of platelet aggregation,regulation of blood flow in kidney and stomach, and regulation ofgastric acid secretion, COX-2 enzyme plays an important role in the painand inflammatory processes. NSAIDs significantly increase clotting timeand can be used for prophylaxis of thromboembolism and myocardialinfarction.

All NSAIDs are relatively medium to strong organic acids with pKa's inthe 3-6 range. Most of them are carboxylic acid derivatives. Acidicgroup is essential for COX inhibitory activity and in physiological pH,all the NSAIDs are ionized. All of them have quite varying hydrophiliclipophilic balance, and these are functions of their aromatic andaliphatic side chains and other heterocyclic variations in theirstructures. Most of the NSAIDs are highly bound to plasma proteins andoften competitively replace other drugs which have similar affinity forplasma proteins. Hence concomitant administration of NSAIDs with othertherapeutic class of drugs must be carefully evaluated to prevent druginteractions. Most of the drugs, due to their acidic carboxyl group, aremetabolized by the mammals in vivo. The major pathway of metabolicclearance of a number of NSAIDs is glucuronidation followed by renalelimination.

Use of acetylsalicylic acid (aspirin) in the prophylaxis of coronaryheart diseases is now well known, and this drug has proved to be alifesaver for a number of patients with myocardial infarction (HeartAttacks). Several additional uses have already been documented foraspirin, for example, it was recently reported in the medical journalLancet (Vol 349, p 1641) that aspirin reduces the risk of stroke inpatients with early warning signs of transient ischemic heart attacks.Pre-eclampsia and fetal growth retardation, both caused by blockages ofthe blood vessels of the placenta, are two of the commonestcomplications of pregnancy—there are millions of cases of pre-eclampsiain the world each year. In a trial involving more than 9000 women in 16countries, a daily dose of 60 mg aspirin reduced the risk ofpre-eclampsia by 13 percent. (Aspirin Foundation website). Aspirin hasalso been shown to be effective in some studies to prevent colon cancer,lung cancer and pancreatic cancer in post-menopausal women. Sinceaspirin can improve blood flow, its usefulness in the treatment ofdiabetes and certain forms of dementia such as Alzheimer's disease arebecoming increasingly clear.

Because of their unique pharmaceutical potential, the NSAIDs haveattracted considerable attention in the press. The primary area ofclinical investigation for the above drugs has been as non-steroidalanti-inflammatory agents, in particular in relation to their applicationto treat patients suffering from pain, arthritis, (Rheumatoid and Osteo)other inflammatory reactions, fever and for the prophylaxis of coronaryheart diseases. These drugs are also used in the treatment of migraineheadaches, menstrual syndromes, back pain and gout.

Despite the very major contribution which NSAIDs have made, difficultieshave been encountered in providing more effective and convenient meansof administration (e.g., galenic formulations, for example, oral dosageform, which are both convenient and for the patient as well as providingappropriate bioavailability and allowing dosaging at an appropriate andcontrolled dosage rate). In addition, there are reported occurrences ofundesirable in vivo side reactions associated with NSAIDs; in particularsevere gastric and duodenal ulcers, mucosal erythema, and edema,erosions, perforations, blood in stool, ulcerative colitis have beenobvious serious impediments to their wider use or application. The dualinjury theory involves NSAID-mediated direct damage, followed by asystemic effect in which prostaglandin synthesis is inhibited. Topicalinjury may also occur as a result of the biliary excretion of activehepatic metabolites and subsequent duodenogastric reflux. (Arthritis andRheumatism 1995; 38(1):5-18) The effects are additive; either topical orsystemic mechanisms alone are sufficient to produce gastro duodenalmucosal damage. Recently, many of the COX-2 inhibitory NSAIDs such asVioxx® and Celebrex® have been removed from the market due to theirundesirable and life threatening side effects.

Moreover, the above mentioned NSAIDs are characteristically highlyhydrophobic and readily precipitate in the presence of even very minoramounts of water, e.g., on contact with the body (e.g., stomach fluids).It is accordingly extremely difficult to provide e.g., oral formulationswhich are acceptable to the patient in terms of form and taste, whichare stable on storage and which can be administered on a regular basisto provide suitable and controlling patient dosaging.

Proposed liquid formulations, e.g., for oral administration of NSAIDs,have heretofore been based primarily on the use of natural gums, likeXanthan, cellulose, citric acid, and lime flavor etc. See e.g., U.S.Pat. No. 5,780,046. Commercially available NSAIDs drink-solution employsincompatible orange color and berry flavor, citric acid, Xanthan Gum,polysorbate 80, pregelatinized starch, glycerin, sodium benzoate, andadditional artificial colors and flavors. Use of the drink solution andsimilar composition as proposed in the art is however accompanied by avariety of difficulties.

Further, the palatability of the known oil based system has provedproblematic. The taste of the known drink-solution is, in particular,unpleasant. Admixture with an appropriate flavored drink, for example,chocolate drink preparation, at high dilution immediately prior toingestion has generally been practiced in order to make regular therapyat all acceptable. Adoption of oil based systems has also required theuse of high ethanol concentrations which in and of itself is inherentlyundesirable, in particular where administration to children is forseen.In addition, evaporation of the ethanol, e.g., from capsules (adopted inlarge part, to meet problems of palatability, as discussed or otherforms (e.g., when opened) results in the development of a NSAIDprecipitate. Where such compositions are presented in, for example, softgelatin encapsulated form presents particular difficulty whichnecessitates packaging of the encapsulated product in an air-tightcomponent, for example, an air-tight blister or aluminum-foil blisterpackage. This in turn renders the product both bulky and more expensiveto produce. The storage characteristics of the aforesaid formulationsare, in addition, far from ideal.

Gastric irritability of the NSAIDs has been a topic of great concern tothe practicing physicians as well as patients. Acute uses of aspirin,fenoprofen, flurbiprofen, indomethacin, ketorolac, meclofenamate,mefanamic acid, and piroxicam produce serious GI side effects. EvenIbuprofen has been shown to cause severe gastric lesions upon long termuse. Gastrointestinal toxicity is the most frequently encountered sideeffect associated with NSAIDs and presents considerable concern.Approximately one half of all hospital admissions for a bleeding ulcerare attributed to the use of NSAIDs, aspirin, or the two taken incombination during the week prior to admission. (Faulkner G, Prichard P,Somerville K, et al. Aspirin and bleeding peptic ulcers in the elderly.Br Med J. 1988; 297:1311-1313). A survey of Tennessee Medicaid patientswho were hospitalized with GI complications showed that patients whoused NSAIDs had approximately a fourfold greater risk for developing GIhemorrhage or peptic ulcer disease than patients not taking NSAIDs.(Griffin M R, Piper J M, Daugherty J R, et al. Nonsteroidalanti-inflammatory drug use and increased risk for peptic ulcer diseasein elderly persons. Ann Intern Med. 1991; 114:257-263). Serious GIevents, according to the FDA, occur in as many as 2% to 4% of patientsper year who are taking continuous NSAID therapy for rheumatoidarthritis. The relative risk of gastric ulcer (4.725), duodenal ulcer(1.1 to 1.6), bleeding (3.8), perforation, and death are all increasedby NSAID use when such patients are compared to those who do not takethese products. In 1989, patients with rheumatoid arthritis hadapproximately 20,000 hospitalizations per year with an estimated cost of$10,000 per stay. (Fries J F, Miller S R, Spitz P W, et al. Toward anepidemiology of gastropathy associated with nonsteroidalanti-inflammatory drug use. J. Gastroenterology. 1989; 96:647-655).

There is also a need for providing some of the NSAIDs in a water solubleform for injection. It is well known that high concentrations of alcoholand tromethamine used to form a salt in the current formulations ofKetorolac are toxic. At present there is no formulation that would allowthe NSAIDs to be in aqueous solution at the concentrations needed due topoor water solubility of the drug.

Beyond all these very evident practical difficulties lies the occurrenceof undesirable side reactions already alluded to, observed employingavailable oral dosage forms.

Several proposals to meet these various problems have been suggested inthe art, including both solid and liquid oral dosage forms. Anoverriding difficulty which has however remained is the inherentinsolubility of the NSAIDs in aqueous media, hence preventing the use ofa dosage form which can contain NSAIDs in sufficiently highconcentration to permit convenient use and yet meet the requiredcriteria in terms of bioavailability, e.g. enabling effective resorptionfrom the stomach or gut lumen and achievement of consistent andappropriately high blood/blood-serum levels.

The present derivatives of NSAIDs overcome the problems describedhereinabove. More specifically, an embodiment of the present inventionis directed to a derivative of NSAID which significantly enhances itssolubility in aqueous solutions, thereby avoiding the need to utilize acarrier, such as ethanol or castor oil when administered as a solution.Moreover, the derivatives of NSAID, in accordance with the presentinvention, do not exhibit the side effects of the prior artformulations. Further, the derivatives of the present invention arealmost completely devoid of gastric irritability upon oraladministration, thereby enhancing significantly the therapeutic index ofthe derivatives tested and their efficacy.

Accordingly, in one aspect, the present invention is directed to aderivative of NSAIDs.

The preferred derivatives of the NSAIDs have the formula

or pharmaceutically acceptable salts thereof; wherein Y is either NH-AAor O-AA and AA is an amino acid residue, in which either an amine groupon the main chain or for those basic acids, on the side chain, if one ispresent (and more preferably on the main chain), or the hydroxyl groupon the side chain of the AA acid residue is reacted with the carboxylicacid group of the NSAIDs.

The present invention is also directed to a pharmaceutical compositioncomprising a therapeutically effective amount of the various amino acidderivatives of the NSAIDs above and a pharmaceutical carrier therefor.

In another embodiment, the present invention is directed to a method oftreating a patient in need of NSAID therapy, which method comprisesadministering to said patient an effective amount of the amino acidderivatives of the NSAIDs of the present invention.

In a further embodiment, the present invention is directed to a methodof enhancing the solubility of NSAID in an aqueous solution comprisingreacting the carboxyl functionality of each of the NSAID with an aminoacid under effective conditions to form a covalent bond between theamino acid and the NSAID and isolating the product thereof.

In a still further embodiment, the present invention is directed to amethod of substantially and in a therapeutically efficacious manner,reducing or eliminating the gastric mucosal damage of NSAIDs whenadministered to a patient which comprises reacting the carboxylfunctionality of each of the NSAID molecule with either the aminefunctionality on the amino acid or hydroxyl function on the amino acidshaving a hydroxy group on the side chain, e.g., Threonine, hydroxyproline, tyrosine, serine and the like, or the amine of those aminoacids having an amino group on the side chain, to form either an amideor ester or an amide covalent bond, respectively, and isolating theproduct thereof and administering said product to the patient.Preferably, the carboxyl functionality of the NSAID is reacted with theamine functionality on the main chain and/or the hydroxy group on theside chains of those amino acids having a hydroxy group on the sidechain.

A. Synthesis of Ibuprofen Amino Acid Derivatives Overview:

The procedure for the synthesis of the L-serine, L-threonine, andL-hydroxyproline esters of Ibuprofen is outlined in Synthetic Sequencesection. The complete procedure and analytical data is given in theExperimental Section. Again, these synthetic schemes are exemplary. Thescheme is applicable for other amino acids in the preparation of theNSAID derivatives of the present invention. In general, (±)-Ibuprofen(4-10 g, in batches) was coupled with the N-benzyloxy/benzyl esterprotected amino acids (1 equivalent) with1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC, 1equivalent) in the presence of a catalytic amount of4-(N,N-dimethyamino)-pyridine (DMAP). Once the reactions were complete,any excess EDC was removed by extraction with water, DMAP was removed byextraction with dilute acid, and Ibuprofen was removed by extractionwith sodium bicarbonate. After drying over sodium sulfate, filtration,and concentration the crude protected amino acid esters of (±)-Ibuprofenwere either used directly or purified by flash chromatography on silicagel to generate the protected esters in good yield (85-95%). The columnchromatography was generally not necessary if a slight excess ofIbuprofen and coupling agent were used, and a thorough extractionprocedure was conducted. The protecting groups were removed byhydrogenation (25-35 psi H₂) in the presence of 10% palladium on carbonand hydrochloric acid. Yields for the deprotection step ranged from70-90%. After filtration and drying, the hydrochloride salts of theserine and threonine esters of (±)-Ibuprofen were purified bycrystallization. The hydrochloride salt of theL-hydroxyproline-Ibuprofen ester was a gel that would notsolidify/crystallize. In this case the hydrogenation was repeatedwithout the use of acid and the neutral compound was purified.

Because the Ibuprofen started as a mixture of enantiomers, the finalproducts were delivered as a mixture of diastereomers except for thethreonine ester. In the case of the threonine ester of Ibuprofen,washing with water, acetone or acetonitrile could readily separate thefinal diastereomeric salts. The insoluble isomer (SPI0016A) wasdetermined to be the active isomer by comparison with an authenticstandard prepared from S-(+)-Ibuprofen. The serine and hydroxyprolineesters of (±)-Ibuprofen could not be readily separated in this fashion.

Synthetic Sequence:

Synthesis of the L-Serine, L-Threonine, and L-Hydroxyproline Esters of(±)-Ibuprofen a) EDC, DMAP, CH₂Cl₂; b) HCl, 10% Pd/C, EtOH c) acetone,d) 10% Pd/C, EtOH Experimental Section

The synthesis of SPI0015, SPI0016 and SPI0017 were conducted in two orthree batches. Reagents mentioned in the experimental section werepurchased at the highest obtainable purity from Sigma-Aldrich, Acros, orBachem, except for solvents, which were purchased from either FisherScientific or Mallinkrodt.

1) Preparation of (±)-Ibuprofen-L-Serine Ester, Hydrochloride (SPI0015)

(±)-Ibuprofen (5.04 g, 24.4 mmole), N-carbobenzyloxy-L-serine benzylester (8.11 g, 24.6 mmole),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC, 4.87g, 25.4 mmole), and 4-(N,N-dimethylamino)-pyridine (DMAP, 0.40 g, 3.27mmole) were dissolved in dichloromethane (150 mL) at room temperature,under an argon atmosphere. After stirring for 22 hours under an argonatmosphere at room temperature, water (100 mL) was added and the layerswere separated. The dichloromethane layer was washed again with water(100 mL) and dried for 1 hour over sodium sulfate (5 g). Afterfiltration and concentration under reduced pressure, the remaining oilwas purified by flash chromatography on silica gel (250 g), eluting withhexanes/ethyl acetate (3:1). The procedure generated the protectedL-serine-(±)-Ibuprofen ester (SPI001501) as a colorless solid (11.4 g,90% yield).

2(S)-Benzyloxycarbonylamino-3-[2(R,S)-(4-isobutyl-phenyl)-propionyloxy]-propionicacid benzyl ester

¹H NMR (300 MHz, CDCl₃): δ=7.40-7.20 (m, 10H), 7.14-7.01 (m, 4H), 5.50(d, ½H, J=8.4 Hz), 5.29 (d, ½H, J=8.4 Hz), 5.11-5.02 (m, 2.5H), 4.90 (d,½H, J=12 Hz), 4.62 (m, 1H), 4.49-4.43 (m, 1H), 4.36-4.32 (m, 1H), 3.59(m, 1H), 2.39-2.35 (m, 2H), 1.78 (m, 1H), 1.42-1.39 (m, 3H), 0.85 (d,6H, J=6.6 Hz).

¹³C NMR (75 MHz, CDCl₃): δ=174.05, 169.19, 169.07, 155.68, 140.73,137.20, 136.12, 135.05, 134.91, 129.44, 128.67, 128.65, 128.60, 128.41,128.33, 128.30, 128.19, 127.19, 127.16, 67.75, 67.32, 64.51, 64.32,53.71, 45.16, 45.02, 30.35, 22.60, 18.27.

The protected Ibuprofen-L-serine ester (22.50 g, 43.4 mmole) wasdissolved in ethanol (200 mL) at room temperature and added to a Parrbottle that contained 10% palladium on carbon (3.86 g, 50% wet) under anitrogen atmosphere. Hydrochloric acid (10 mL 37% HCl in 30 mL water)was added and the nitrogen atmosphere was replaced with hydrogen gas (25psi). After 4 hours of shaking, the palladium catalyst was removed byfiltration through celite. The ethanol/water was removed under reducedpressure. The remaining white solids were washed with water (25 mL),acetone (20 mL) and dried under high vacuum (4 hours at 88° C.). Theexperiment produced (±)-Ibuprofen-L-serine ester, hydrochloride SPI0015(11.3 g, 80% yield) as a colorless solid.

2(S)-Amino-3-[2(R,S)-(4-isobutylphenyl)-propionyloxy]-propionic acid,hydrochloride; ((R,S)-Ibuprofen-L-Serine ester, hydrochloride)

¹H NMR (300 MHz, DMSO): δ=8.92 (br s, 3H), 7.22 (t, 2H, J=7.5 Hz), 7.10(d, 2H, J=7.5 Hz), 4.56 (m, 1H), 4.37-4.20 (m, 2H), 3.83 (q, 1H, J=6.9Hz), 2.41 (d, 2H, J=6.9 Hz), 1.80 (m, 1H), 1.41 (d, 3H, J=6.9 Hz), 0.85(d, 6H, J=6.9 Hz).

¹³C NMR (75 MHz, DMSO): δ=173.36, 173.32, 168.08, 168.04, 139.70,128.96, 129.92, 127.20, 127.05, 62.47, 51.59, 51.49, 44.28, 44.00,43.90, 29.68, 22.28, 18.70, 18.42.

HPLC Analysis:

99.13% purity; rt=3.133 min; Luna C18 5 u column (sn 167917-13); 4.6×250mm; 254 nm; 50% ACN/50% TFA buffer (0.1%); 35 C; 20 ul inj.; 1 ml/min; 1mg/mL sample size; sample dissolved in mobile phase.

CHN Analysis:

calc.: C, 58.27; H, 7.33; N, 4.25. found: C, 58.44; H, 7.46; N, 4.25.

Melting point: 169.5-170.5° C.

2a) Preparation and Separation of (±)-Ibuprofen-L-Threonine Ester,Hydrochloride (SPI0016A and SPI0016B)

(±)-Ibuprofen (4.15 g, 20.11 mmole), N-carbobenzyloxy-L-threonine benzylester (6.90 g, 20.11 mmole),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC, 3.95g, 20.6 mmole), and 4-(N,N-dimethylamino)-pyridine (DMAP, 0.25 g, 2.0mmole) were dissolved in dichloromethane (50 mL) at room temperature,under an argon atmosphere. After stirring for 19 hours, thedichloromethane layer was washed with water (50 mL), 5% hydrochloricacid (2×25 mL), water (25 mL), saturated sodium bicarbonate (2×25 mL),and water (50 mL). After drying for one hour over sodium sulfate (5 g),filtration, and concentration under reduced pressure, the remaining oilwas used without further purification. The procedure generated theprotected L-threonine-(±)-Ibuprofen ester (SPI001601) as a light yellowoil (10.2 g, 95.3% yield), which solidified on standing.

2(S)-Benzyloxycarbonylamino-3-[2(R,S)-(4-isobutyl-phenyl)-propionyloxy]-butyricacid benzyl ester

¹H NMR (300 MHz, CDCl₃): δ=7.40-7.15 (m, 10H), 7.14-7.01 (m, 4H),5.48-5.25 (m, 2H), 5.11-5.01 (m, 3H), 4.90 (d, ½H, J=12 Hz), 4.68 (d,½H, J=12 Hz), 4.48 (m, 1H), 3.60-3.48 (m, 1H), 2.39 (m, 2H), 1.79 (m,1H), 1.42-1.35 (m, 3H), 1.27 (d, 1.5H, J=6.6 Hz), 1.17 (d, 1.5H, J=6.6Hz), 0.85 (m, 6H).

¹³C NMR (75 MHz, CDCl₃): δ=173.32, 169.70, 169.30, 156.55, 140.75,137.38, 137.22, 136.14, 135.07, 134.99, 129.45, 129.41, 128.65, 128.39,128.22, 127.21, 127.14, 70.97, 70.70, 67.81, 67.66, 67.53, 57.83, 45.19,30.39, 22.61, 18.57, 18.30, 17.18, 16.87.

The protected Ibuprofen-L-threonine ester (10.15 g, 19.0 mmole) wasdissolved in warm ethanol (150 mL) and added to a Parr bottle thatcontained 10% palladium on carbon (3.4 g, 50% wet) under a nitrogenatmosphere. Hydrochloric acid (6 mL 37% HCl in 20 mL water) was addedand the nitrogen atmosphere was replaced with hydrogen gas (30 psi).After 3 hours of shaking, the palladium catalyst was removed byfiltration through celite (30 g). The ethanol/water was removed underreduced pressure. The experiment produced (±)-Ibuprofen-L-threonineester, hydrochloride (SPI0016A and SPI0016B, 6.4 g, 97% crude yield) asa colorless solid. The crude mixture of diastereomers was stirred inacetone (200 mL) for 2 hours at room temperature under an argonatmosphere. After 2 hours the solids (2.84 g, SPI0016A) were filtered.The filtrate (SPI0016B, 3.0 g) was concentrated under reduced pressure.

1.) Purification of SPI0016A (Active Isomer):

After 3 batches of the S-Ibuprofen-L-threonine ester (SPI0016A) had beencompleted, the batches were combined (8.78 g total) and crystallizedthree times from DIUF (“deionized ultrafiltered”) water (100 mL). Eachtime a small amount of zwitterion was generated. In order to regeneratethe salt, the solid generated (from each crystallization) was dissolvedin 1% hydrochloric acid in ethanol (3 mL 37% hydrochloric acid in 100 mLethanol). The ethanol solution was then concentrated under reducedpressure at room temperature. After the third crystallization andregeneration procedure, the salt (5.6 g) was stirred in acetonitrile(100 mL) for 44 hours at room temperature, under an argon atmosphere.The salt was then filtered and dried under high vacuum at 50-55° untilthe weight was constant (5.5 g).

2(S)-Amino-3(R)-[2(S)-(4-isobutyl-phenyl)-propionyloxy]-butyric acid;(S-Ibuprofen-L-Threonine Ester, Hydrochloride, Active Isomer)

¹H NMR (300 MHz, DMSO): δ=8.76 (br s, 3H), 7.19 (d, 2H, J=8.1 Hz), 7.11(d, 2H, J=8.1 Hz), 5128 (dq, 1H, J=6.3, 3.6 Hz), 4.14 (q, 1H, J=3.6 Hz),3:80 (q, 1H, J=7.2 Hz), 2.41 (d, 2H, J=7.2 Hz), 1.80 (m, 1H), 1.37 (d,3H, J=7.2 Hz), 1.21 (d, 3H, J=6.3 Hz), 0.85 (d, 6H, J=6.6 Hz).

¹³C NMR (75 MHz, DMSO): δ=172.66, 168.24, 139.68, 137.24, 128.95,126.97, 67.98, 55.35, 44.23, 43.83, 29.66, 22.24, 18.52, 16.47.

CHN Analysis:

calc.: C, 59.38; H, 7.62; N, 4.07. found: C, 59.17; H, 7.63; N, 4.04.

HPLC Analysis:

98.28% purity; r.t.=6.951 min.; 60% TFA (0.1%)/40% acetonitrile; 1mL/min; 37.5 C; Luna C18, 3 u column (SN 167917-13), 4.6×250 mm; 22 ulinjection.

Optical rotation: +24.5°

Melting Point: 189-190° C.

2) Purification of SPI0016B (Inactive Isomer):

After 3 batches of the R-Ibuprofen-L-threonine ester (SPI0016B) had beencompleted, the batches were combined (9.02 g total) and crystallizedfrom DIUF (deionized ultra filtered) water (50 mL). A small amount ofzwitterion was generated during the crystallization. In order toregenerate the salt, the solid generated was dissolved in 1%hydrochloric acid in ethanol (3 mL 37% hydrochloric acid in 100 mLethanol). The ethanol solution was then concentrated under reducedpressure at room temperature. The remaining salt (5.93 g) wascrystallized three times from hot toluene (100 mL) with the addition ofa small amount on acetone (1 mL). The salt was then filtered and driedunder high vacuum at room temperature until the weight was constant (5.1g).

2(S)-Amino-3(R)-[2(R)-(4-isobutyl-phenyl)-propionyloxy]-butyric acid;(R-Ibuprofen-L-Threonine Ester, Hydrochloride, Inactive Isomer)

¹H NMR (300 MHz, DMSO): δ=8.82 (br s, 3H), 7.23 (d, 2H, J=7.8 Hz), 7.10(d, 2H, J=7.8 Hz), 5.27 (m, 1H), 4.18 (m, 1H), 3.80 (q, 1H, J=7.2 Hz),2.41 (d, 2H, J=7.2 Hz), 1.81 (m, 1H), 1.41 (d, 3H, J=6.9 Hz), 1.34 (d,3H, J=6.3 Hz), 0.85 (d, 6H, J=6.3 Hz).

¹³C NMR (75 MHz, DMSO): δ=72.56, 168.08, 139.64, 136.98, 128.84, 127.14,68.8, 55.29, 44.28, 29.69, 22.28, 18.24, 16.41.

CHN Analysis:

calc.: C, 59.38; H, 7.62; N, 4.07. found: C, 59.30; H, 7.60; N, 4.05.

HPLC Analysis:

98.43% purity; r.t.=6.19 min.; 60% TFA (0.1%)/40% acetonitrile; 1mL/min; 37.5 C; Luna C18, 3 u column (SN 167917-13), 4.6×250 mm; 22 ulinjection.

Optical Rotation: +10.4°

Melting Point: 176-177° C.

2b) Preparation of the S-(+)-Ibuprofen-L-Threonine Ester, HydrochlorideStandard (SPI0016S)

S-(+)-Ibuprofen (2.0 g, 9.69 mmole), N-carbobenzyloxy-L-threonine benzylester (3.25 g, 9.91 mmole),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC, 1.90g, 9.91 mmole), and 4-(N,N-dimethylamino)-pyridine (DMAP, 0.12 g, 1.0mmole) were dissolved in dichloromethane (25 mL) at room temperature,under an argon atmosphere. After stirring for 4 hours, thedichloromethane layer was washed with water (25 mL), 5% hydrochloricacid (25 mL), saturated sodium bicarbonate (2×25 mL), and water (25 mL).After drying for one hour over sodium sulfate (5 g), filtration, andconcentration under reduced pressure, the remaining oil was used withoutfurther purification. The procedure generated the protectedS-(+)-Ibuprofen-L-threonine ester (SPI001601S) as a light yellow oil(5.01 g, 98% yield), which solidified on standing.

2(S)-Benzyloxycarbonylamino-3-[2(R,S)-(4-isobutyl-phenyl)-propionyloxy]-butyricacid benzyl ester

¹H NMR (300 MHz, CDCl₃): δ=7.35-7.23 (m, 10H), 7.10 (d, 2H, J=7.8 Hz),7.05 (d, 2H, J=7.8 Hz), 5.48-5.25 (m, 2H), 5.17-5.01 (m, 4H), 4.50 (dd,1H, J=9.6, 1.8 Hz), 3.50 (q, 1H, J=7.2 Hz), 2.40 (d, 2H, J=7.2 Hz), 1.80(m, 1H), 1.37 (d, 3H, J=7.2 Hz), 1.17 (d, 3H, J=6.3 Hz), 0.86 (d, 6H,J=6.6 Hz).

¹³C NMR (75 MHz, CDCl₃): δ=173.29, 169.69, 156.51, 140.68, 137.21,136.08, 135.06, 129.40, 128.70, 128.66, 128.57, 128.38, 128.24, 127.14,70.70, 67.80, 67.53, 57.87, 45.19, 45.11, 30.39, 22.61, 18.57′, 16.87.

The protected S-(+)-Ibuprofen-L-threonine ester (5.0 g, 9.40 mmole) wasdissolved in warm ethanol (100 mL) and added to a Parr bottle thatcontained 10% palladium on carbon (1.0 g, 50% wet) under a nitrogenatmosphere. Hydrochloric acid (1 mL 37% HCl in 10 mL water) was addedand the nitrogen atmosphere was replaced with hydrogen gas (32 psi).After 2 hours of shaking, the palladium catalyst was removed byfiltration through celite (30 g). The ethanol/water was removed underreduced pressure. The experiment produced S-(+)-Ibuprofen-L-threonineester, hydrochloride (SPI0016S, 2.8 g, 85% crude yield) as a colorlesssolid. The salt was stirred in acetone (50 mL) for 3 hours at roomtemperature under an argon atmosphere. After 3 hours the solids (2.24 g,69% yield) were filtered and dried under high vacuum at roomtemperature, until the weight was constant.

2(S)-Amino-3(R)-[2(S)-(4-isobutyl-phenyl)-propionyloxy]-butyric acid;(S-Ibuprofen-L-Threonine Ester, Hydrochloride, Active Isomer)

¹H NMR (300 MHz, DMSO): δ=8.76 (br s, 3H), 7.19 (d, 2H, J=8.1 Hz), 7.11(d, 2H, J=8.1 Hz), 5.28 (dq, 1H, J=6.3, 3.6 Hz), 4.14 (q, 1H, J=3.6 Hz),3.80 (q, 1H, J=7.2 Hz), 2.41 (d, 2H, J=7.2 Hz), 1::80 (m, 1H), 1.37 (d,3H, J=7.2 Hz), 1.21 (d, 3H, J=6.3 Hz), 0.85 (d, 6H, J=6.6 Hz).

¹³C NMR (75 MHz, DMSO): δ=172.66, 168.24, 139.68, 137.24, 128.95,126.97, 67.98, 55.35, 44.23, 43.83, 29.66, 22.24, 18.52, 16.47.

HPLC Analysis:

98.28% purity; r.t.=6.951 min.; 60% TFA (0.1%)/40% acetonitrile; 1mL/min; 37.5 C; Luna C18, 3 u column (SN 167917-13), 4.6×250 mm; 22 ulinjection.

Optical rotation: +26.5°

Melting Point: 189-190° C.

3) Preparation of the (±)-Ibuprofen-L-hydroxyproline ester (SPI0017).

(±)-Ibuprofen (5.10 g, 24.7 mmole), N-carbobenzyloxy-L-hydroxyprolinebenzyl ester (8.80 g, 24.7 mmole),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC, 5.10g, 26.0 mmole), and 4-(N,N-dimethylamino)-pyridine (DMAP, 0.30 g, 2.40mmole) were dissolved in dichloromethane (100 mL) at room temperature,under an argon atmosphere. After stirring for 24 hours under an argonatmosphere at room temperature, water (100 mL) was added and the layerswere separated. The dichloromethane layer was washed again with water(100 mL), 5% sodium bicarbonate (2×50 mL) and dried for 1 hour oversodium sulfate (5 g). After filtration and concentration under reducedpressure, the remaining oil was used without further purification. Theprocedure generated the protected (±)-Ibuprofen-L-hydroxyproline ester(SPI001701) as a light yellow oil (11.5 g, 85% yield).

4(R)-[2-(4-Isobutyl-phenyl)-propionyloxy]-pyrrolidine-2(S)-carboxylicacid; ((R,S)-Ibuprofen-L-hydroxyproline ester)

¹H NMR (300 MHz, CDCl₃): δ=7.33-7.02 (m, 14H), 5.25-4.95 (m, 5H),4.51-4.19 (m, 1H), 3.75-3.50 (m, 3H), 2.40 (d, 2H, J=6.9 Hz), 2.15 (m,1H), 1.81 (m, 1H), 1.44 (d, 3H, J=7.0 Hz), 0.87 (d, 6H, J=6.6 Hz).

¹³C NMR (75 MHz, CDCl₃): δ=173.99, 171.93, 171.72, 154.68, 154.15,140.70, 137.23, 137.04, 136.23, 135.44, 135.23, 129.41, 128.59, 128.47,128.35, 128.19, 128.08, 127.89, 127.02, 72.86, 72.16, 67.40, 67.18,67.09, 58.12, 57.83, 52.66, 52.49, 52.13, 45.15, 36.63, 35.67, 32.07,30.33, 29.23, 22.90, 22.58, 18.36.

The protected Ibuprofen-L-hydroxyproline ester (11.40 g, 43.4 mmole) wasdissolved in ethanol (150 mL) at room temperature and added to a Parrbottle that contained 10% palladium on carbon (2.73 g, 50% wet) under anitrogen atmosphere. The nitrogen atmosphere was replaced with hydrogengas (34 psi). After 5 hours of shaking, the palladium catalyst wasremoved by filtration through celite. The ethanol was removed underreduced pressure. The remaining white solids (6.60 g) were washed withDIUF water (50 mL), diethyl ether (50 mL) and dried under high vacuumuntil the weight was constant. The experiment produced(±)-Ibuprofen-L-hydroxyproline ester SPI0017 (5.64 g, 84% yield) as acolorless solid.

4(R)-[2-(4-Isobutyl-phenyl)-propionyloxy]-pyrrolidine-2(S)-carboxylicacid; ((R,S)-Ibuprofen-L-hydroxyproline ester)

¹H NMR (300 MHz, CDCl₃): δ=7.22 (d, 2H, J=7.2 Hz), 7.09 (d, 2H, J=7.2Hz), 5.27 (m, 1H), 4.40 (t, 0.5H, J=7 Hz), 4.24 (t, 0.5H, J=9Hz), 3.75(m, 1H), 3.61 (m, 1H), 3.28 (d, 0.5H, J=13 Hz), 3.15 (d, 0.5H, J=13 Hz),2.42-2.10 (m, 4H), 1.78 (m, 1H), 1.40 (br t, 3H, J=6 Hz), 0.82 (d, 6H,J=6 Hz). (mixture of diastereomers)

¹³C NMR (75 MHz, CDCl₃): δ=173.28, 173.23, 168.98, 139.88, 137.33,137.23, 129.12, 127.26, 127.17, 72.58, 57.60, 57.50, 50.24, 50.12,44.34, 44.15, 34.31, 34.16, 29.77, 22.34, 18.43, 18.23. (mixture ofdiastereomers)

HPLC Analysis:

100% purity; r.t.=5.35, 5.22 min.; 55% TFA (0.1%), 45% ACN; 1 mL/min;32.3 C, Luna C18, serial #188255-37; 20 ul inj.

CHN Analysis:

calc.: C, 67.69; H, 7.89; N, 4.39. found: C, 67.47; H, 7.87; N, 4.30.

Melting Point: 198-199° C.

Efficacy (Anti Nociceptive Potential) of Synthesis of the L-Serine,L-Threonine, and L-Hydroxyproline Esters of (±)-Ibuprofen by EmployingAcetylcholine Induced Abdominal Constriction Method in Male Albino Mice:

The present study was conducted to evaluate the efficacy of L-serine,L-threonine, and L-hydroxyproline esters of (±)-Ibuprofen taking intoaccount the antagonizing property on acetylcholine induced writhe as anindex in albino mice. Ibuprofen (racemic mixture) and ibuprofen (S)-(+)served as reference controls.

Different new formulations of ibuprofen and reference controls viz.,ibuprofen (racemic mixture) and ibuprofen (S)-(+) were administered bygavage to male albino mice (Swiss strain), using 5% (v/v) Tween 80 inmilli Q water as the vehicle. The study was conducted at two dose levelsviz. 50 mg and 100 mg/kg body weight along with a vehicle control group.At each dose level 10 animals were used. All the doses were expressed asibuprofen molar equivalents. The doses used as well as the molarequivalents are presented below.

TABLE 5 Formulation: Molar Equivalent: Formulation Molar equivalentS-(+)-Ibuprofen- 0.833 units are equivalent L-threonine ester to 1 unitof Ibuprofen (±)-Ibuprofen- 1.6 units are equivalent L-serine ester to 1unit of Ibuprofen (±)-Ibuprofen-L- 1.55 units are equivalenthydroxyproline ester to 1 unit of Ibuprofen

TABLE 6 Test Item: Group: Dose(mg/kg): Equivalent wt. Of the test item:Equivalent Dose (mg per kg) weight of the [in terms of Test item TestItem Group Ibuprofen] [mg/kg] Vehicle Vehicle control 0.0 — GroupS-(+)-Ibuprofen-L- Test Group 1 50.0 41.65 threonine ester Test Group 2100.0 83.30 (±)-Ibuprofen-L- Test Group 3 50.0. 80.0 serine ester TestGroup 4 100.0 160.0 (Ibuprofen S) (±)-Ibuprofen-L- Test Group 5 50.077.5 hydroxyproline ester Test Group 6 100.0 155.0 Ibuprofen (racemicTest Group 7 50.0 50.0 mixture) Test Group 8 100.0 100.0 Ibuprofen S +Test Group 9 50.0 25.0 Test Group 10 100.0 50.0

The efficacy in terms of antagonizing effect on acetylcholine inducedsingle writhe at two dose levels −50.0 and 100.0 mg/kg for the threeformulations and reference controls are presented below.

TABLE 7 Test Item: Group: Dose (mg/kg): Number of animals showingabsence of single writhe (out of 10) Number of animals showing absenceof single writhe (number of Dose (mg animals per dose = 10) per kg) Onehour Three [in terms of after hours after Test Item Group Ibuprofen]dosing dosing Vehicle Vehicle control 0.0 0 0 S-(+)- Low dose 50.0 1 0Ibuprofen-L- High dose 100.0 3 0 threonine ester (±)- Low dose 50.0 4 2Ibuprofen-L- High dose 100.0 6 4 serine ester (±)- Low dose 50.0 5 4Ibuprofen-L- High dose 100.0 7 7 hydroxyproline ester Ibuprofen Low dose50.0 4 2 (racemic High dose 100.0 6 6 mixture) Ibuprofen S + Low dose50.0 5 1 High dose 100.0 6 6

Statistical analysis employing Chi-square test procedure did not showany statistically significant difference among the formulations incomparison to reference control, while comparing the number of animalsnot showing writhe in each groups, as the respective “p” was found to begreater than 0.05, the level of significance.

From clinical observation based on the number of animals not showingwrithes due to administration of acetylcholine,(±)-Ibuprofen-L-hydroxyproline ester was found to be more effective inantagonizing the acetylcholine induced writhe when compared to otherformulations and Ibuprofen (racemic) and Ibuprofen (S)-(+).

TABLE 8 Summary of Efficacy of L-serine, L-threonine, and L-hydroxyproline esters of (±)-Ibuprofen, Ibuprofen (racemic mixture) andIbuprofen (S)-(+)-Based on Antagonizing Property of AcetylcholineInduced Writhe in Albino Mice Number of animals showing Dose absence ofsingle writhe (number (mg/kg) of animals per dose = 10) [in terms of Onehour after Three hours Ibuprofen] Test Item dosing after dosing 50 mg/kgVehicle control 0 0 S-(+)-Ibuprofen-L- 1 0 threonine ester(±)-Ibuprofen-L- 4 2 serine ester (±)-Ibuprofen-L- hydroxyproline 5 4ester Ibuprofen 4 2 (racemic mixture) Ibuprofen (S)-(+) 5 1

TABLE 9 100 mg/kg Vehicle control 0 0 S-(+)-Ibuprofen- 3 0 L-threonineester (±)-Ibuprofen-L- 6 4 serine ester (±)-Ibuprofen-L- 7 7hydroxyproline ester Ibuprofen 6 6 (racemic mixture) Ibuprofen (S)-(+) 66

The data were subjected to statistical analysis employing Chi-squaretest procedure for evaluating the efficacy of the new formulations incomparison to the reference controls. The tests did not show anystatistically significant difference among the formulations incomparison to reference control, while comparing the number of animalsnot showing writhe in each groups, as the respective “p” was found to begreater than 0.05, the level of significance.

The data is also summarized in FIGS. 1 and 2. From clinical observationsand bar diagram for comparative efficacy (FIGS. 1 and 2), based on thenumber of animals not showing writhes due to administration ofacetylcholine, (±)-Ibuprofen-L-hydroxyproline ester was found to be moreeffective in antagonizing the acetylcholine induced writhe when comparedto other formulations and Ibuprofen (racemic) and Ibuprofen (S)-(+).

Conclusion

The present study was conducted to evaluate the relative efficacy of newformulations of ibuprofen. For this the antagonizing property of newformulations on acetylcholine writhes was taken as an index to determinethe relative efficacy of the formulations. Ibuprofen (racemic mixtureand ibuprofen (S)-(+) served as reference controls. The study wasconducted at two dose levels (50.0 and 100.0 mg/kg) along with a vehiclecontrol group.

The efficacy in terms of antagonizing effect of acetylcholine inducedsingle writhe at two dose levels −50.0 and 100.0 mg/kg for the threeformulations and reference controls are presented below.

TABLE 10 Test Item: Group: Dose (mg/kg): No. of animals showing absenceof single writhe (out of 10) Number of animals showing absence of singleDose (mg writhe (number of animals per kg) per dose = 10) [in terms ofOne hour Three hours Test Item Group Ibuprofen] after dosing afterdosing Vehicle Vehicle 0.0 0 0 control S-(+)-Ibuprofen- Low dose 50.0 10 L-threonine High dose 100.0 3 0 ester (±)-Ibuprofen-L- Low dose 50.0 42 serine ester High dose 100.0 6 4 (±)-Ibuprofen-L- Low dose 50.0 5 4hydroxyproline High dose 100.0 7 7 ester Ibuprofen Low dose 50.0 4 2(racemic mixture) High dose 100.0 6 6 Ibuprofen (S)- Low dose 50.0 5 1(+) High dose 100.0 6 6

Statistical analysis employing Chi-square test procedure did not showany statistically significant difference among the formulations incomparison to reference control, while comparing the number of animalsnot showing writhe in each groups, as the respective “p” was found to begreater than 0.05, the level of significance.

However from clinical observation based on the number of animals notshowing writhes due to administration of acetylcholine(±)-Ibuprofen-L-hydroxyproline ester was found to be more effective inantagonizing the acetylcholine induced writhe when compared to otherformulations and Ibuprofen (racemic) and Ibuprofen (S)-(+).

Gastric Mucosal Irritation Potential of L-Serine, L-Threonine, andL-Hydroxyproline Esters of (±)-Ibuprofen in Fasted Male Albino RatsSUMMARY

The present study was conducted to determine the relative potential ofnew formulations of ibuprofen (L-serine, L-threonine, andL-hydroxyproline esters of (±)-Ibuprofen) to cause gastric mucosalirritation/lesions in fasted male albino rats. Ibuprofen (racemicmixture) and Ibuprofen(S)-(+) served as reference controls.

Different new formulations of ibuprofen and ibuprofen (racemic mixture)and ibuprofen(S)-(+) were administered by gavage to fasted male albinorats (Wistar strain), using 5% solution of Tween 80 in milli Q water asthe vehicle. The study was conducted at two dose levels viz. 200 mg and300 mg/kg body weight along with a vehicle control group. At each doselevel 5 animals were used. All the doses were expressed as ibuprofen(racemic mixture) molar equivalents. The doses used as well as the molarequivalents were presented below.

TABLE 11 Formulation: Molar Equivalent Formulation Molar equivalentS-(+)-Ibuprofen- 0.833 units are equivalent to L-threonine ester 1 unitof Ibuprofen (±)-Ibuprofen- 1.60 units are equivalent to L-serine ester1 unit of Ibuprofen (±)-Ibuprofen- 1.55 units are equivalent toL-hydroxyproline ester 1 unit of IbuprofenThe various groups used are tabulated hereinbelow:

TABLE 12 Test item: group: Dose (mg/kg) Equivalent wt. Equivalent Dose(mg weight of per kg) the [in terms of Test item Test item GroupIbuprofen] [mg/kg] Vehicle Vehicle control 0.0 — GroupS-(+)-Ibuprofen-L- Test Group 1 200.0 0.0 threonine ester Test Group 2300.0 166.6 (±)-Ibuprofen-L-serine Test Group 1 200.0 249.9 ester TestGroup 2 300.0 320.0 (±)-Ibuprofen-L- Test Group 1 200.0 480.0hydroxyproline ester Test Group 2 300.0 310.0 Ibuprofen (racemic TestGroup 1 200.0 465.0 mixture) Test Group 2 300.0 300.0 Ibuprofen (S)- (+)Test Group 1 200.0 100.0 Test Group 2 300.0 150.0

The rats were fasted for a period of 18 to 22 hours before dosing. Thetest item was administered as a single dose by gavage. Three hours afterdrug administration, the animals were killed humanely by CO₂ gasinhalation. The stomach was dissected out and observed for

-   -   the quantity of mucous exudate,    -   degree of hyperemia and thickening of stomach wall,    -   hemorrhagic spots (focal or diffuse), nature of hemorrhages        (petechial or ecchymotic) along with the size and    -   perforations or any other lesions

The observations on gastric mucosal irritation of animals of variousgroups were summarized below:

TABLE 13 Test item: Group: Dose (mg/kg): Observation Doe mg/kg (as perTest Item Group ibuprofen) Observation Vehicle control Vehicle control0.0 None of the animals Group showed any evidence of gastric mucosalirritation S-(+)-Ibuprofen- Test Group 1 200.0 None of the dosedL-threonine ester animals showed any evidence of gastric mucosalirritation Test Group 2 300.0 None of the dosed animals showed anyevidence of gastric mucosal irritation. (±)-Ibuprofen-L- Test Group 1200.0 None of the dosed serine ester animals showed any evidence ofgastric mucosal irritation. Test Group 2 300.0 None of the dosed animalsshowed any evidence of gastric mucosal irritation (±)-Ibuprofen-L- TestGroup 1 200.0 None of the dosed hydroxyproline animals showed any esterevidence of gastric mucosal irritation Test Group 2 300.0 None of thedosed animals showed any evidence of gastric mucosal irritationIbuprofen Test Group 1 200.0 Gastric mucosal (racemic mixture)irritation was observed in one animal out of 5 animals dosed. Test Group2 300.0 Gastric mucosal irritation was observed in two animals out of 5animals dosed. Ibuprofen (S)- Test Group 1 200.0 Gastric mucosal (+)irritation was observed in all the 5 animals dosed. Test Group 2 300.0Gastric mucosal irritation was observed in three animals out of 5animals dosed.

The results of the present study showed that none of the formulations ofibuprofen had caused any evidence of irritation of gastric mucosa infasted male albino rats of male sex at the two dose levels tested (200mg and 300 mg/kg body weight). In contrast, both ibuprofen (racemicmixture) and ibuprofen (S)-(+) had caused irritation of gastric mucosaat the two dose levels tested. Further ibuprofen(S)-(+) was found to bemore gastric mucosal irritant than ibuprofen (racemic mixture).

28-Day Chronic Toxicity Studies with S(±) Ibuprofen L-Threonin Ester inRats

Chronic toxicity of S(±) Ibuprofen-L-Threonine ester was comparedagainst a vehicle, (+/−) racemic Ibuprofen and (+/−) racemicIbuprofen-L-Hydroproline ester. Test species used was Swiss Albino Mice,both male and female with body weight range of 18-27 gms. Randomizationwas done by the method of stratified randomization procedure using SASsoftware program (Version 8.2) with stratification by bodyweight.

TABLE 4 NUMBER OF ANIMAL NUMBERS GROUP TEST ITEM ANIMALS Female MaleVehicle Control Vehicle 10 01 to 05 06 to 10 Test Group 1 Formulation 110 11 to 15 16 to 20 of Ibuprofen Test Group 2 Formulation 5 10 21 to 2526 to 30 of Ibuprofen Reference Test Ibuprofen USP 10 31 to 35 36 to 40Group

The test doses are expressed as Ibuprofen molar equivalents:

TABLE 5 Dose (mg per kg) Equivalent [in terms of weight of the Test ItemGroup Ibuprofen] Test item [mg] Vehicle Vehicle 0.0 0.0 ControlL-Threonine ester of Test Group 1 200.0 334.0 S(+) IbuprofenL-Hydroxyproline ester Test Group 2 200.0 310.0 of Racemin IbuprofenIbuprofen USP Reference (Racemic mixture) Control 200.0 200.0

The duration of dosing was 28 days. All the animals were daily testeduntil the end of the study for the presence/absence of clinical symptomsof toxicity. Cage side observations included changes in the skin, eyes,posture, gait, respiration and behavior pattern. The incidence oftwitching, tremors, convulsions, salivation, diarrhea and death if any,were also recorded.

Animals Exposed to Different Doses of the Test Substance did notIndicate any Symptoms of Toxicity (Table 16).

TABLE 16 Summary of Clinical Symptoms of Toxicity in Albino Mice PeriodGROUP of Signs (mg/kg body Symptoms Animal in days weight) of toxicitySex Numbers From-to Mortality Vehicle No symptoms Female 01 to 05 0-28Nil Control (0.0) of toxicity (No Treatment) were observed No symptomsMale  6 to 10 0-28 Nil of toxicity were observed Test Group 1 Nosymptoms Female 11 to 15 0-28 Nil (Ibuprofen of toxicity S + T) wereobserved (334.0 mg/kg) No symptoms Male 16 to 20 0-28 Nil of toxicitywere observed Test Group 2 No symptoms Female 21 to 25 0-28 1/5(Ibuprofen HP) of toxicity were observed (310.0 mg/kg) No symptoms Male26 to 30 0-28 Nil of toxicity were observed Reference No symptoms Female31 to 35 0-28 3/5 Control 3 of toxicity (Ibuprofen were observed USP)(200.0 mg/kg) No symptoms Male 36 to 40 0-28 1/5 of toxicity wereobservedIn the above table, S+T Ibuprofen refers to S(+)Ibuprofen-L-ThreonineEster.

Death Record

Ibuprofen HP

Animal no. 23 (female animal) died on 21 day of dosing.

Positive Control Group.

Animal no. 31 (female animal) died on 23 day of dosing.

Animal no. 32 (female animal) died on 21 day of dosing.

Animal no. 33 (female animal) died on 24 day of dosing.

Animal no. 40 (male animal) died on 10 day of dosing.

While there were no cage side specific toxicity were noted, surprisingly40% of the animals receiving racemic ibuprofen died, only 10% of therats receiving Hydroxyproline ester of Ibuprofen did not complete thefull course, and even more surprisingly, none of the animals in theS(+)Ibuprofen-L-Threonine ester group died. The averageincrease/decrease in body weight and the percentage change of bodyweight of the surviving animals in various groups are shown below:

TABLE 17 Average Change Percentage Change In body (No of TreatmentWeight (gms) animals survived) Vehicle 4.65 19.97 (10) S(+)IbuprofenThreonine Ester −0.68 −3.61 (10) Ibuprofen Hydroxy- Proline Ester 1.435.75 (9) Racemic Ibuprofen 2.97 12.54 (6)

While there was increase in body weight in treatments with racemicIbuprofen and Ibuprofen Hydroxyproline ester, both group havemortalities, with currently marketed Ibuprofen showing more mortalitythan Hydroxyproline ester. Hence all the amino acid esters are farsuperior to Ibuprofen racemic mixture or the active S(+)Ibuprofen.However, the best product so far seems to be S(+)Ibuprofen-L-ThreonineEster, making it one of the ideal candidates to be advanced to humantrials.

Human Clinical Trials with S(+)Ibuprofen-L-Threonine Ester:

Determination of the analgesic and anti-inflammatory effects ofS(+)Ibuprofen-L-Threonine Ester in three human volunteers was conductedas follows:

Two male, age 49 and 50 having severe headache took 1 capsule containingS(+)Ibuprofen-L-Threonine Ester manufacturing and formulation under GMPconditions. The capsule contents were equivalent to 200 mg of racemicIbuprofen. Relief from headache was reported after 15 min, and completeabsence of any pain from headache was reported at the end of 1 hour,which lasted for another 12 hours.

Two males age 49 and 51 took 1 capsule each containingS(+)IbuprofenL-Threonine ester for arthritic knee pain, which wasperceptible. After 12 hours, both volunteers reported significantreduction is the pain associated with their right knee. Suchamolearation of pain was further sustained for another 24 hours.

Pharmacokinetics of Ibuprofen in Human Volunteers:

Based upon preliminary analgesic and anti-inflammatory response from the4 volunteers, Ibuprofen racemic drug was compared againstS(+)Ibuprofen-L-Threonine ester at 200 mg equivalent dose. Theplasma-concentration time profile in 6 volunteers, whereS(+)Ibuprofen-L-Threonine ester concentrations were plotted againstracemic ibuprofen concentrations in plasma for each volunteers.

Based upon the results of comparative bioavailablity of racemicIbuprofen versus Ibuprofen released from S(+)Ibuprofen-L-Threonine esterit is clear that only very small amount of Ibuprofen is released intactinto human blood stream. This is due to the fact thatS(+)Ibuprofen-L-threonine ester does not act as a derivative ofIbuprofen, instead the Threonine ester had intact activity.

The overall bioavailability of Ibuprofen from Ibuprofen Racemic mixtureof 200 mg and equivalent dose of S(±)Ibuprofen-L-Threonine ester areshown in the table below:

TABLE 18 Volunteer AUC1 AUC2 % Availability 1 69197.618 893.226 1.3 241861.277 1978.925 4.7 3 73121.747 940.133 1.3 4 38993.502 2101.642 5.45 34567.246 1657.496 4.8 6 66710.152 925.000 1.4

As used herein, the term AUC refers to area under the curve. In theabove table, AUC1 represents the cumulative area under the plasmaconcentration time curve following oral administration of 200 mg ofracemic ibuprofen to human volunteers, and AUC2 represents thecumulative area under the plasma concentration time curve following oraladministration of 200 mg ibuprofen equivalence ofS(+)Ibuprofen-L-Threonine ester. The third column in the above tableshows relative bioavailability of Ibuprofen in human plasma after oraladministration of S(+)Ibuprofen-L-Threonine ester at equivalent doses.This clearly demonstrates that any activity seen in human volunteers isnot due to release of any significant amounts of S(+)Ibuprofen in to theplasma after oral ingestion of the Threonine ester.

The reason that S(+)Ibuprofen advanced to human pharmacokinetic studieswas due to surprising results of lack of toxicity in 28-day chronicadministration in rats compared to Ibuprofen, or Hydroxyproline ester ofIbuprofen. Furthermore, earlier studies indicated that S(+)Ibuprofen ishighly toxic to gastric mucosa of rats (see table X above). Similarresults were also shown in various studies elsewhere, for example, otherinvestigators compared S(+) and R(−) enantiomers of ibuprofen in maleWistar rats. At 40 mg/kg dose, microscopic evaluation of the GI tissuesamples revealed significance difference in GI toxicity caused by S(+)than R(−) Ibuprofen, consistent with current inventors results(Janjikhel, R K, Bricker, J D, Borochovitz, D, Adeyeye, C M,Stereoselective Disposition of Sustained Release Microspheres ofIbuprofen Enantiomers in Rats: II, Acute Gastrointestinal Toxicity. DrugDelivery, Vol 6, No. 3, August 1999, pp 163-170). HoweverS(+)Ibuprofen-L-Threonine ester was GI sparing, and had no toxicity.

Furthermore, other authors have claimed that R(−)Ibuprofen may becapable of inhibiting both therapeutic and toxic effects ofS(+)Ibuprofen (Kaehler, S T, Phleps, W, Hesse, E. Dexibuprofen:Pharmacology, therapeutic uses and safety. Infammopharmacology, Vol 11No. 4-6, 2003, pp 371-383). This is also consistent with currentinventor's observation, since in acute GI toxicity studies racemicibuprofen was somewhat less toxic than S(+)Ibuprofen.

Similarly, Rainsford K D, in Pharmacology and Toxicology of Ibuprofen.In: Rainsford, K D, ed. Ibuprofen, A critical bibliographic Review.London, Taylor and Francis, 2000, states that competition between theenantiomers of ibuprofen for prostaglandin production in vitro wasevident, and that inhibition of binding of S(+) ibuprofen by R(−)ibuprofen in the racemic mixture contributed to the GI tolerance of theracemate.

However, what is not known in the previous art is that an amino acidderivative, e.g., an ester of S(+)Ibuprofen would also be nontoxic. Forexample, one trained in the art of such derivative pharmacology, wouldhave concluded that as S(+)Ibuprofen is highly toxic to the GI mucosa,and since S(+)Ibuprofen will be released from S(+)Ibuprofen-L-Threonineester by the esterase enzymes in GI tract, and by the action ofpancreatin enzyme in the duodenum, it was expected that there will notbe any reduction in toxicity. However, the current inventor surprisinglynoted that S(+)Ibuprofen-L-Threonine ester is significantly andcompletely non-toxic to GI mucosa in the rats tested.

In spite of no ibuprofen appearing in the human plasma after oraladministration of S(+)Ibuprofen-L-Threonine ester, significant analgesicand anti-inflammatory response was seen in the 2 volunteers each testedtwice. This is novel since it appears, that S(+)Ibuprofen-L-threonineester is not a derivative, and it may have intact pharmacologicalactivity. This is also not anticipated from the prior art.

While less effectiveness was seen in rat model withS(+)Ibuprofen-L-Threonine ester, the overriding factors that demonstratethat this drug is more suitable for human treatment of various diseasessuch as arthritis etc., is due to the fact that on chronic toxicitytrials none of the animal died in this drug group. Furthermore, notoxicity either gastric or whole animal (as determined by number ofsurviving rats), was observed in the rat model for S(+)-IbuprotenL-Threonine ester. Further, there is no toxicity potential in thesystemic circulation in human subjects. The S(+)Ibuprofen-L-Threonineester exhibits pharmacological actions such as analgesic,anti-inflammatory and anti-pyretic properties when administered tohumans.

Synthesis of Ketoprofen Derivatives Overview:

The procedure for the synthesis of the L-threonine esters of Ketoprofenis outlined in Synthetic Sequence section. The complete procedure andanalytical data is given in the Experimental Section. In general,(±)-Ketoprofen (5 g) was coupled with N-boc-L-threonine t-butyl ester (1equivalent) with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide,hydrochloride (EDC, 1 equivalent) in the presence of a catalytic amountof 4-(N,N-dimethyamino)-pyridine (DMAP). Once the reaction was complete,any excess EDC was removed by extraction with water, DMAP was removed byextraction with dilute acid, and Ketoprofen was removed by extractionwith sodium bicarbonate. After drying over sodium sulfate, filtration,and concentration, the crude protected L-threonine-(±)-Ketoprofen waspurified by flash chromatography on silica gel to generate the protectedL-threonine ester in good yield (98%). The protecting groups wereremoved by treatment with 2M hydrochloric acid in diethyl ether tocleave the boc group, followed by treatment with trifluoroacetic acid toremove the t-butyl ester. After drying, the mixture ofL-threonine-R,S(±)-Ketoprofen esters was separated by crystallizationfrom acetonitrile. The hydrochloride salt of theL-threonine-S(+)-Ketoprofen ester preferentially precipitated fromacetonitrile. A sample of an optically pure standard was preparedstarting with S(+)-ketoprofen for comparison. After drying and analysis,a sample of L-threonine-S(+)-Ketoprofen ester, hydrochloride (1.75 g)separated from the mixture was shipped to Signature Pharmaceuticals,Inc. for testing.

The L-serine and L-hydroxyproline esters of (±)-Ketoprofen were alsoprepared in the same manner. Attempts to separate the mixture ofKetoprofen diastereomers by crystallization, using a variety ofsolvents, failed to separate the mixture. Since only partial separationcould be seen by HPLC analysis, the esters were purified as the mixtureof diastereomers before final analysis. The preparation and finalanalytical data for the serine and hydroxyproline esters are alsoincluded.

Synthetic Sequence:

Synthesis of the L-Threonine Esters of (±)-Ketoprofen a) EDC, DMAP,CH₂Cl₂; b) HCl (2M); c) TFA; d) ACN (crystallization) ExperimentalSection

The synthesis of SPI0018A was conducted in a single batch. Reagentsmentioned in the experimental section were purchased at the highestobtainable purity from Sigma-Aldrich, Acros, or Bachem, except forsolvents, which were purchased from either Fisher Scientific orMallinkrodt.

Preparation and Separation of S(+)-Ketoprofen-L-Threonine Ester,Hydrochloride (SPI0018A).

(±)-Ketoprofen (5.32 g, 20.92 mmol), N-t-butylcarbonyl-L-threoninet-butyl ester (Boc-Thr-OtBu, 5.17 g, 18.72 mmol, prepared by theliterature method), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide,hydrochloride (EDC, 4.0 g, 20.9 mmol), and4-(N,N-dimethylamino)-pyridine (DMAP, 0.22 g) were dissolved indichloromethane (50 mL) at room temperature, under an argon atmosphere.After stirring for 5 hours, the dichloromethane layer was washed withwater (50 mL), 5% hydrochloric acid (2×25 mL), water (25 mL), saturatedsodium bicarbonate (2×25 mL), and water (50 mL). After drying for onehour over sodium sulfate (5 g), filtration, and concentration underreduced pressure, the remaining oil (10.3 g) was purified by columnchromatography on silica gel (150 g), eluting with hexanes/ethyl acetate(2:1). After combining the product containing fractions, concentrationand drying under high vacuum, the procedure generated the protectedL-threonine-(±)-Ketoprofen ester (SPI001801) as a clear oil (9.42 g, 98%yield).

3-[2(R,S)-(3-Benzoyl-phenyl)-propionyloxy]-2(S)-tert-butoxycarbonylamino-butyricacid tert-butyl ester: (mix of diastereomers)

¹H NMR (300 MHz, CDCl₃): δ=7.83-7.42 (m, 9H), 5.43 (dd, 1H, J=13.2, 6.9Hz), 5.10 (dd, 1H, J=20.7, 9.3), 4.29 (t, 1H, J=11.7 Hz), 3.75 (q, 1H,J=7.2 Hz), 1.50-1.42 (m, 19.5H), 1.30-1.18 (m, 4.5H).

¹³C NMR (75 MHz, CDCl₃): δ=.196.18, 172.62, 172.55, 168.85, 168.58,155.81, 140.33, 140.23, 137.86, 137.39, 132.46, 132.42, 131.54, 131.38,130.00, 129.31, 129.13, 129.02, 128.54, 128.27, 82.50, 82.37, 80.05,71.38, 71.22, 57.59, 57.52, 45.46, 45.31, 28.40, 27.98, 27.84, 18.54,18.48, 17.19, 16.84.

The protected (R,S)-Ketoprofen-L-threonine ester (9.42 g, 18.41 mmol)was dissolved in dichloromethane (25 mL) under an argon atmosphere, atroom temperature. Anhydrous hydrochloric acid in diethyl ether (2M, 25mL) was added to the solution and the mixture was allowed to stir for 17hours at room temperature. The mixture was concentrated under reducedpressure. The remaining foam (8.2 g) was dissolved in a mixture ofdichloromethane (10 mL) and trifluoroacetic acid (20 mL). After stirringat room temperature for 6.5 hours the solution was concentrated underreduced pressure. Toluene (25 mL) was added to the remaining oil and themixture was concentrated a second time. A mixture of ethanol (20 mL) andanhydrous hydrochloric acid in diethyl ether (2M, 20 mL) was added andthe solution was concentrated a third time. After drying under highvacuum for 2 hours at room temperature, the experiment produced(±)-Ketoprofen-L-threonine ester, hydrochloride (mixture ofdiastereomers, 7.11 g, 98% crude yield) as an off-white solid. The crudemixture of diastereomers (7.0 g) was crystallized 3 times fromacetonitrile (200 mL). After the third crystallization, the remainingwhite solid was dried under high vacuum at 50° C. until the weight wasconstant (4 hours). The experiment produced L-threonine-S(+)-Ketoprofenester, hydrochloride SPI0018A (2.2 g, 30% yield from SPI001801).

2(S)-Amino-3(R)-[2(S)-(3-benzoyl-phenyl)-propionyloxy]-butyric acid,hydrochloride (L-threonine-S(+)-Ketoprofen ester, hydrochloride)

¹H NMR (300 MHz, DMSO): δ=14.08 (br s, 1H), 8.72 (br s, 3H), 7.74-7.51(m, 9H), 5.29 (t, 1H, J=4.5 Hz), 4.16 (m, 1H), 3.97 (q, 1H, J=6.3 Hz),1.42 (d, 3H, J=6.9 Hz), 1.23 (d, 3H, J=6.3 Hz).

¹³C NMR (75 MHz, DMSO): δ=195.34, 172.26, 168.21, 140.42, 137.05,136.74, 132.66, 131.66, 129.48, 128.73, 128.49, 128.30, 68.23, 55.31,44.00, 18.44, 16.45.

CNN Analysis:

calc.: C, 61.30; H, 5.66; N, 3.57. found: C, 61.02; H, 5.58; N, 3.58.

HPLC Analysis:

98.28% purity; r.t.=25.14 min.; 55% DIUF water (0.1% TFA)/45% methanol;1 mL/min; 36.4 C; Luna C18, 5 u column (serial #211739-42), 4.6×250 mm;20 ul injection.

Optical rotation: +27.0° (20 C, 174.4 mg/10 mL ethanol, 589 nm)

Melting Point: 166-167° C.

Preparation of the S-(+)-Ketoprofen-L-Threonine Ester, HydrochlorideStandard

(+)-Ketoprofen (1.87 g, 7.74 mmol), N-t-butylcarbonyl-L-threoninet-butyl ester (Boc-Thr-OtBu, 2.25 g, 8.14 mmol, prepared by theliterature method), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide,hydrochloride (EDC, 1.65 g, 8.60 mmol), and4-(N,N-dimethylamino)-pyridine (DMAP, 0.1 g) were dissolved indichloromethane (25 mL) at room temperature, under an argon atmosphere.After stirring for 4 hours, the dichloromethane layer was washed withwater (25 mL). After drying for one hour over sodium sulfate (5 g),filtration, and concentration under reduced pressure, the remaining oilwas used without purification. The procedure generated the protectedL-threonine-(+)-Ketoprofen ester as a clear oil (4.01 g, ˜100% yield).

¹H NMR (300 MHz, CDCl₃): δ=7.81-7.42 (m, 9H), 5.43 (m, 1H), 5.10 (d, 1H,J=9.3), 4.29 (d, 1H, J=9.6 Hz), 3.75 (q, 1H, J=7.2 Hz), 1.50-1.42 (m,21H), 1.18 (d, 3H, J=6.3 Hz).

¹³C NMR (75 MHz, CDCl₃): δ=196.4, 172.79, 168.99, 155.94, 140.44,137.99, 137.51, 132.59, 131.50, 130.13, 129.31, 129.25, 129.15, 128.66,128.40, 82.68, 80.24, 71.37, 57.71, 45.43, 28.53, 28.10, 18.99, 16.96.

The protected (S)-Ketoprofen-L-threonine ester (3.92 g, 7.66 mmol) wasdissolved in anhydrous hydrochloric acid in diethyl ether (2M, 50 mL)and stirred for 17 hours at room temperature. The mixture wasconcentrated under reduced pressure. The remaining foam (3.4 g) wasdissolved in a mixture of dichloromethane (20 mL) and trifluoroaceticacid (20 mL). After stirring at room temperature for 6.5 hours thesolution was concentrated under reduced pressure. Toluene (25 mL) wasadded to the remaining oil and the mixture was concentrated a secondtime. A mixture of ethanol (20 mL) and anhydrous hydrochloric acid indiethyl ether (2M, 20 mL) was added, and the solution was concentrated athird time. After drying under high vacuum for 2 hours at roomtemperature, the experiment produced S(+)-Ketoprofen-L-threonine ester,hydrochloride (3.05 g crude) as an off-white solid. The crude materialwas stirred with acetone (50 mL) for 2 hours at room temperature underan argon atmosphere. The remaining white solid was filtered and driedunder high vacuum at 50° C. until the weight was constant (4 hours). Theexperiment produced L-threonine-S(+)-Ketoprofen ester, hydrochloride(2.04 g, 67% yield).

¹H NMR (300 MHz, DMSO): δ=14.08 (br s, 1H), 8.72 (br s, 3H), 7.74-7.51(m, 9H), 5.29 (t, 1H, J=4.5 Hz), 4.16 (m, 1H), 3.97 (q, 1H, J=6.3 Hz),1.42 (d, 3H, J=6.9 Hz), 1.23 (d, 3H, J=6.3 Hz).

¹³C NMR (75 MHz, DMSO): δ=195.34, 172.26, 168.21, 140.42, 137.05,136.74, 132.66, 131.66, 129.48, 128.73, 128.49, 128.30, 68.23, 55.31,44.00, 18.44, 16.45.

HPLC Analysis:

99.43% purity; r.t.=25.14 min.; 55% DIUF water (0.1% TFA)/45% methanol;1 mL/min; 36.4 C; Luna C18, 5 u column (serial #211739-42), 4.6×250 mm;20 ul injection.

Optical rotation: +27.1° (20 C, 177.8 mg/10 mL ethanol, 589 nm)

Melting Point: 166-167° C.

Preparation of the (±)Ketoprofen-L-Serine Ester, Hydrochloride

(±)-Ketoprofen (7.30 g, 28.7 mmol), N-t-butylcarbonyl-L-serine t-butylester (Boc-Ser-OtBu, 7.50 g, 28.7 mmol, prepared by the literaturemethod), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride(EDC, 5.5 g, 28.7 mmol), and 4-(N,N-dimethylamino)-pyridine (DMAP, 0.12g) were dissolved in dichloromethane (50 mL) at room temperature, underan argon atmosphere. After stirring for 3 hours, the dichloromethanelayer was washed first with water (50 mL), then 5% hydrochloric acid(2×25 mL), then water (25 mL) again, saturated sodium bicarbonate (2×25mL), and water (50 mL) a third time. After drying for one hour oversodium sulfate (5 g), filtration, and concentration under reducedpressure, the remaining foam was used without purification. Theprocedure generated the protected L-serine-(±)-Ketoprofen ester as aclear foam (13.72 g, 96% yield).

3-[2(R,S)-(3-Benzoyl-phenyl)-propionyloxy]-2(S)-tert-butoxycarbonylamino-propionicacid tert-butyl ester

¹H NMR (300 MHz, CDCl₃): δ=7.77-7.38 (m, 9H), 5.29 (d, ½H, J=6.9 Hz),5.13 (d, ½H, J=6.9 Hz), 4.44-4.30 (m, 3H), 3.78 (q, 1H, J=7 Hz), 1.50(d, 3H, J=7 Hz), 1.39 (m, 18H).

¹³C NMR (75 MHz, CDCl₃): δ=. 196.13, 173.37, 168.37, 154.99, 140.40,137.89, 132.50, 131.44, 130.01, 129.25, 129.14, 128.53, 128.29, 82.72,80.03, 65.22, 64.91, 53.62, 53.40, 45.29, 28.41, 28.02, 27.91, 18.59,18.50.

The protected (R,S)-Ketoprofen-L-serine ester (13.6 g, 127.31 mmol) wasdissolved in anhydrous hydrochloric acid in diethyl ether (2M, 100 mL)under an argon atmosphere, at room temperature. The mixture was allowedto stir for 23 hours at room temperature when dichloromethane was added(100 mL). After 48 hours, the mixture was concentrated under reducedpressure. The remaining light yellow foam (9.0 g) was dissolved in amixture of dichloromethane (200 mL) and DIUF water (50 mL). After mixingat room temperature, the layers were separated. The dichloromethanelayer was acidified with 2N hydrochloric acid in ether (5 mL), driedover sodium sulfate (10 g) filtered and concentrated under reducedpressure. The remaining foam (6.4 g) was stirred with dichloromethane(40 mL) for 30 minutes at room temperature under an argon atmosphere.Diethyl ether was added (20 mL) and the mixture was allowed to stir for2 hours at room temperature. After 2 hours, the solids were filtered anddried under high vacuum at room temperature until a constant weight wasobtained. The experiment produced L-serine-R,S(±)-Ketoprofen ester,hydrochloride (2.5 g, 22% yield).

2(S)-Amino-3-[2(R,S)-(3-benzoyl-phenyl)-propionyloxy]-propionic acid,hydrochloride

H NMR (300 MHz, DMSO): δ=8.79 (br s, 3H), 8.72 (br s, 3H), 7.76-7.54 (m,9H), 4.57 (m, 1H), 4.42-4.28 (m, 2H), 4.01 (m, 1H), 1.46 (d, 3H, J=6Hz).

¹³C NMR (75 MHz, DMSO): δ=195.33, 172.92, 168.01, 167.96, 140.50,140.39, 136.97 (d), 136.75, 132.66, 131.93 (d), 129.55, 128.65 (d),128.49 (d), 62.18, 51.35 (d), 44.07, 18.62, 18.41.

HPLC Analysis

98.99% purity; r.t.=9.205 min. (broad peak); 55% DIUF water (0.1%TFA)/45% methanol; 1 mL/min; 36.4 C; Luna C18, 5 u column (serial#211739-42), 4.6×250 mm; 20 ul injection.

CHN Analysis:

calc.: C, 60.40; H, 5.34; N, 3.71. found: C, 60.15; H, 5.32; N, 3.72.

Melting Point: 116-120° C. (uncorrected)

Preparation of the (±)Ketoprofen-L-Hydroxyproline Ester, Hydrochloride

(±)-Ketoprofen (6.70 g, 26.3 mmol),N-t-butylcarbonyl-trans-L-hydroxyproline-t-butyl ester (Boc-Hyp-OtBu,7.40 g, 25.7 mmol, prepared by the literature method),1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC, 5.25g, 27.3 mmol), and 4-(N,N-dimethylamino)-pyridine (DMAP, 0.10 g) weredissolved in dichloromethane (50 mL) at room temperature, under an argonatmosphere. After stirring for 3.5 hours, the dichloromethane layer waswashed first with water (50 mL), then, 5% hydrochloric acid (2×25 mL),then water (25 mL), then saturated sodium bicarbonate (2×25 mL), andfinally water (50 mL). After drying for one hour over sodium sulfate (5g), filtration, and concentration under reduced pressure, the remaininglight green oil (13.30 g) was purified by column chromatography onsilica gel (120 g), eluting with heptane/ethyl acetate (2:1). Aftercombining the fractions containing product, concentration under reducedpressure and drying under high vacuum, the procedure generated theprotected L-hydroxyproline-(±)-Ketoprofen ester as a clear oil (5.50 g,41% yield).

4(R)-[2(R,S)-(3-Benzoyl-phenyl)-propionyloxy]-pyrrolidine-1,2(S)-dicarboxylicacid di-tert-butyl ester

¹H NMR (300 MHz, CDCl₃): δ=7.77-7.38 (m, 9H), 5.29 (d, ½H, J=6.9 Hz),5.13 (d, ½H, J=6.9 Hz), 4.44-4.30 (m, 3H), 3.78 (q, 1H, J=7 Hz), 1.50(d, 3H, J=7 Hz), 1.39 (m, 18H).

¹³C NMR (75 MHz, CDCl₃): δ=196.25, 173.43 (d), 171.46 (d), 153.66 (d),140.35, 138.00, 137.47, 132.55, 131.38, 130.05, 129.13, 128.67, 128.30,81.55, 80.37 (d), 73.31, 72.48, 58.56, 51.86 (d), 45.43, 36.68 (d),28.49, 28.18, 18.60.

The protected (R,S)-Ketoprofen-L-hydroxyproline ester (3.30 g, 6.31mmol) was dissolved in anhydrous hydrochloric acid in diethyl ether (2M,20 mL) under an argon atmosphere at room temperature. After 72 hours,the mixture was concentrated under reduced pressure. The remaining lightyellow foam (2.6 g) was dissolved in a mixture of dichloromethane (50mL) and DIUF water (10 mL). After mixing at room temperature, the layerswere separated. The dichloromethane layer was acidified with 2Nhydrochloric acid in ether (5 mL) dried over sodium sulfate (5 g)filtered and concentrated under reduced pressure. The remaining foam (2g) was stirred with diethyl ether (20 mL) for 30 minutes at roomtemperature under an argon atmosphere. The solids were filtered anddried under high vacuum at room temperature until a constant weight wasobtained. The experiment produced L-hydroxyproline-R,S(±)-Ketoprofenester, hydrochloride (1.2 g, 48% yield).

4(R)-[2(R,S)-(3-Benzoyl-phenyl)-propionyloxy]-pyrrolidine-2(S)-carboxylicacid, hydrochloride

H NMR (300 MHz, DMSO): δ=10.25 (br s, 2H), 7.73-7.53 (m, 9H), 5.29 (brm, 1H), 4.38 (t, ½H, J=8.1 Hx), 4.26 (t, ½H, J=9Hz), 3.95 (m, 1H), 3.60(m, 1H), 3.28 (d, ½H, J=13 Hz), 3.16 (d, ½H, J=12 Hz), 2.37-2.20 (m,2H), 1.45 (m, 3H).

¹³C NMR (75 MHz, DMSO): δ=195.38, 172.78, 172.73, 169.16, 140.50,140.41, 137.08, 136.77, 132.67, 132.01, 131.89, 129.52, 128.78, 128.50,128.50, 72.87 (d), 57.60, 57.52, 50.16 (d), 44.30, 44.20, 34.26, 34.15,18.43, 18.25.

HPLC Analysis:

99.99% purity; r.t.=7.842 and 7.689 min. (broad double peak); 55% DIUFwater (0.1% TFA)/45% methanol; 1 mL/min; 36.4 C; Luna C18, 5 u column(serial #211739-42), 4.6×250 mm; 20 ul injection.

CHN Analysis:

calc.: C, 62.45; H, 5.49; N, 3.47. found: C, 61.78; H, 5.56; N, 3.62.

Melting Point: 170-173° C. (uncorrected).

Synthesis of Keotorlac-L-Threonine Ester and Human Trials Overview:

The procedure for the synthesis of the L-threonine ester of Ketorolac isoutlined in Synthetic Sequence section. The complete procedure andanalytical data is given in the Experimental Section. In general,(±)-Ketorolac was extracted from the tromethamine salt (10 g) andcoupled with N-boc-L-threonine t-butyl ester (1 equivalent) with1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDCI) inthe presence of a catalytic amount of 4-(N,N-dimethylamino)-pyridine(DMAP). The crude protected L-threonine-(±)-Ketorolac ester was purifiedby flash chromatography. The protecting groups were removed by treatmentwith trifluoroacetic acid. The mixture of L-threonine-R,S(±)-Ketorolacester salts was separated by crystallization from acetonitrile/acetone.A sample of S(−)-Ketorolac L-threonine ester, hydrochloride (2.1 g)separated from the mixture was shipped to Signature for testing.

Synthetic Sequence:

Synthesis of the L-Threonine Esters of (±)-Ketorolac

a) AcOH/H₂O, CH₂Cl₂; b) EDC, DMAP, CH₂Cl₂; c) TFA; d) HCl, ethanol; e)ACN-acetone (crystallization).

Experimental Section:

The synthesis of SPI0031A was conducted in a single batch. The procedurewas later repeated to ensure reproducibility. Reagents mentioned in theexperimental section were purchased at the highest obtainable purityfrom Cayman Chemical, Sigma-Aldrich, Acros, or Bachem, except forsolvents, which were purchased from either Fisher Scientific orMallinkrodt.

Preparation and Separation of S(−)-Ketorolac L-Threonine Ester,Hydrochloride (SPI0031A)

(±)-Ketrolac tromethamine salt (10 g, Cayman Chemical) was dissolved inwater (100 mL), acetic acid (20 mL), and dichloromethane (50 mL). Aftermixing for ten minutes, the layers were separated and the water fractionwas extracted two additional times with dichloromethane (50 mL). Thedichloromethane fractions were combined, dried over sodium sulfate,filtered, concentrated, and dried under high vacuum at room temperatureuntil a constant weight was obtained. The procedure generated(±)-Ketrolac (6.78 g, 100% yield) as an off-white solid.

¹H NMR (300 MHz, CDCl₃): δ=9.62 (1H, br s), 7.80 (2H, d, J=6.9 Hz),7.55-7.42 (3H, m), 6.84 (1H, d, J=4.0 Hz), 6.15 (1H, d, J=4.0 Hz),4.62-4.41 (2H, m), 4.10 (1H, dd, J=8.4, 5.7 Hz), 2.97-2.75 (2H, m).

¹³C NMR (75 MHz, CDCl₃): δ=185.25, 176.69, 142.04, 139.05, 131.61,129.02, 128.26, 127.31, 125.43, 103.63, 47.77, 42.64, 31.20.

(±)-Ketrolac (6.80 g, 26.6 mmol), N-tert-butylcarbonyl-L-threoninetert-butyl ester (Boc-Thr-OtBu, 7.33 g, 26.6 mmol, prepared by theliterature method), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide,hydrochloride (EDCI, 5.50 g, 28.6 mmol), and4-(N,N-dimethylamino)-pyridine (DMAP, 0.10 g) were dissolved indichloromethane (75 mL) at room temperature, under an argon atmosphere.After stirring for 6 hours, the dichloromethane solution was washedfirst with water (50 mL), then with saturated sodium bicarbonate 50 mL),and finally with water (50 mL) again. After drying the dichloromethanesolution for one hour over sodium sulfate (10 g), filtration, andconcentration under reduced pressure, the remaining brown oil (14.55 g)was purified by column chromatography on silica gel (250 g), elutingwith heptane/ethyl acetate (1:1). After combining the fractionscontaining product, concentration and drying under high vacuum, theprocedure generated the protected L-threonine-(±)-Ketorolac ester(SPI003101) as light brown solid foam (13.53 g, 99.2% yield).

¹H NMR (300 MHz, CDCl₃): δ=7.80 (2H, m), 7.55-7.42 (3H, m), 6.81 (1H,m), 6.08 (1H, m), 5.47 (1H, m), 5.17 (1H, m), 4.60-4.34 (3H, m), 4.01(1H, m), 2.90-2.70 (2H, m), 1.48-1.32 (21H, m).

¹³C NMR (75 MHz, CDCl₃): δ=184.85, 169.91, 168.91, 155.84, 141.91,139.12, 131.41, 128.86, 128.13, 127.18, 124.99, 103.56, 103.13, 82.76,80.23, 72.15, 72.00, 57.64, 47.62, 42.71, 42.53, 32.02, 31.11, 28.44,28.02, 17.03, 14.33.

The protected (±)-Ketorolac L-threonine ester SPI003101 (13.50 g, 26.33mmol) was dissolved in trifluoroacetic acid (50 mL) under an argonatmosphere, at room temperature. The mixture was allowed to stir for 7hours at room temperature under an argon atmosphere. The brown solutionwas concentrated under reduced pressure and dried under high vacuum atroom temperature until a constant weight was achieved. The remainingbrown solid (10.2 g) was stirred in acetone (250 mL) at room temperaturefor 3 hours. The white precipitate that formed was filtered and driedunder high vacuum. The remaining solid (5.70 g) was dissolved in aminimal amount of DIUF water (5-10 mL) and a 1:1 mixture ofacetonitrile-acetone (100 mL) was added drop-wise over 1 hour whilestirring at room temperature. After the addition was complete, themixture was stored for 2 hours at room temperature. The whiteprecipitate that formed was filtered and dried under high vacuum at roomtemperature to a constant weight. The white solid (3.0 g) was purified afinal time by dissolving in DIUF water (5 mL). Most of the water wasremoved under reduced pressure to generate a thin, clear oil. Acetone(100 mL) was added to the oil in a drop-wise fashion over 30 minuteswhile stirring under an argon atmosphere. The mixture was stored for 3hours at −10° C. The precipitate was filtered and dried under highvacuum at room temperature until the weight was constant. The experimentproduced S(−)-Ketorolac-L-threonine ester, hydrochloride SPI0031A (2.32g, 22.4% yield based on SPI003101, 98.12% purity by HPLC) as a whitesolid.

¹H NMR (300 MHz, DMSO): δ=8.80 (3H, br s), 7.73 (2H, d, J=7.5 Hz),7.61-7.46 (3H, m), 6.77 (1H, d, J=3.9 Hz), 6.16 (1H, d, J=3.9 Hz), 5.33(1H, m), 4.42-4.22 (4H, m), 2.76 (2H, m), 1.35 (3H, d, J=6.6 Hz).

¹³C NMR (75 MHz, DMSO): δ=183.41, 169.64, 168.19, 142.32, 138.59,131.44, 128.35, 128.26, 126.22, 124.27, 103.18, 68.84, 55.31, 47.34,41.83, 30.18, 16.59.

CHN Analysis:

calc.: C, 58.09; H, 5.39; N, 7.13, Cl, 9.02 (C₁₉H₂₁ClN₂O₅). found: C,58.61, H, 5.26, N, 7.10, Cl, 8.16.

HPLC Analysis:

98.12% purity, r.t.=19.617 min, sample dissolved in DIUF water/ACN, 50%DIUF water (0.1% TFA)/50% ACN, Gemini C18 (#262049-2), 5 u, 250×4.6 mm,1 mL/min., 37° C., 20 uL inj. vol., SPD-10Avp, chl-210 nm.

Specific rotation: −108 deg (25° C., 52.5 mg/5 mL water, 589 nm)

Melting Point: 155-157° C. (decomposed)

Large negative specific rotation is consistent with the S(−)Ketorolacmoiety.

Human Clinical Trial:

S(−)Ketorolac-L-Threonine ester capsules were filled using dextrose asthe filler. The Ketorolac-L-Threonine ester dose was comparable toracemic Ketorolac Tromethamine tablets. For example, 13 mg ofS(−)Ketorolac-L-Threonine ester was roughly equivalent to 13 mg ofracemic Ketorolac tromethamine in tablet and/or capsule form.

A Female patient (age 72) suffering from severe ankolysing spodolytis,arthritis and other inflammatory joint problems was under treatment withIndomethacin, 25 mg twice daily dose. In order the evaluate theanalgesic activity of S(−)Ketorolac-L-Threonine ester, the patient waswithdrawn from Indomethacin. After 24 hours, the pain returned and shewas administered with 13 mg of S(−)Ketorolac L-Threonine ester, once inthe morning and once in the evening. This was repeated for 5 days.During the entire period, the patient demonstrated lack of pain, nogastric irritation symptoms, or other side effects.

About 3 months later, the same above female volunteer repeated theexperiment. This time, after she went off the indomethacin, only onedose of 13 mg of S(−)Ketorolac-L-Threonine ester was administered. Afterthe 2^(nd) day, she complained of the pain resurfacing, and the dose wasthen increased on the morning of the 3^(rd) day to twice daily of 13 mgeach. Beginning the 3^(rd) day she indicated of lack of any pain. Thistreatment was continued for another 3 days of two capsules of 13 mgeach. On the morning of the 6^(th) day, she was switched back toIndomethacin. This study showed that in this particular volunteer, 13 mgtwice daily was the appropriate dose to alleviate her severe pain.

C. Amino Acid Derivatives of Aspirin Overview:

The procedure for the synthesis of the L-serine, L-threonine, andL-hydroxyproline esters of acetylsalicylic acid is outlined in SyntheticSequence section and is exemplary for other amino acids. The completeprocedure and analytical data is given in the Experimental Section. Ingeneral, acetylsalicyloyl chloride (10 g-25 g, in batches) was coupledwith the N-benzyloxy/benzyl ester protected amino acids in the presenceof pyridine. Once the reactions were completed (24 to 48 hours at roomtemperature), the mixture was poured into ice-cold 2N hydrochloric acid.The dichloromethane fraction was then washed with sodium bicarbonate,water and brine. After drying over sodium sulfate, filtration, andconcentration, the crude protected amino acid esters of acetylsalicylicacid were purified by flash chromatography on silica gel. The proceduregenerated the protected amino acid esters of acetylsalicylic acid inyields ranging from 68% to 95%. The protecting groups were removed byhydrogenation (20 psi H₂) in the presence of 10% palladium on carbon.The amino acid esters of acetylsalicylic acid were extracted from thepalladium catalyst using water. The solution containing the product usesconcentrated, and dried. The final compounds was washed with solvent(water, dioxane, acetonitrile, and/or dichloromethane) until pure anddried under high vacuum until a constant weight was achieved.

Synthetic Sequence

Synthesis of the L-serine, L-threonine, and L-hydroxyproline esters ofacetylsalicylic acid

a) pyridine, CH₂Cl₂; b) 10% Pd/C, EtOH, EtOAc.

Experimental Section:

The synthesis of SPIB00101, SPIB00102 and SPIB00103 was conducted in oneor two batches. Reagents mentioned in the experimental section werepurchased at the highest obtainable purity from Lancaster,Sigma-Aldrich, Acros, or Bachem, except for solvents, which werepurchased from either Fisher Scientific or Mallinkrodt.

1) SPIB00102 2-O-Acetylsalicylic acid (2S,3R)-(−)-threonine ester

A mixture of N-carbobenzyloxy-L-threonine benzyl ester (Z-Thr-OBzl,21.77 g, 63.40 mmole) and pyridine (25 mL) in anhydrous dichloromethane(500 mL) was cooled in an ice bath while under a nitrogen atmosphere.Acetylsalicyloyl chloride (17.63 g, 88.76 mmole) was added, and themixture was allowed to warm to room temperature and stir overnight.After 24 hours, the mixture was poured into ice-cold 2N hydrochloricacid (400 mL). After mixing, the layers were separated and thedichloromethane fraction was washed first with water (500 mL), thensaturated sodium bicarbonate solution (500 mL), then water (500 mL),then brine (500 mL) and dried over sodium sulfate (25 g). Afterfiltration, concentration under reduced pressure, and drying under highvacuum, the remaining yellow oil (35.43 g) was purified by flashchromatography on silica gel (300 g, 0.035-0.070 mm, 6 nm porediameter), eluting with hexanes/ethyl acetate (3:1). After concentrationof the product containing fractions under reduced pressure and dryingunder high vacuum until the weight was constant, the experiment producedthe protected acetylsalicylic-L-threonine ester SPIB0010201 (28.1 g, 88%yield) as a colorless oil.

¹H NMR (300 MHz, CDCl₃): δ=7.74 (1H, d, J=7.5 Hz), 7.51 (1H, dt, J=7.5,1.5 Hz), 7.34-7.17 (11H, m), 7.06 (1H, d, J=7.2 Hz), 5.62 (2H, m), 5.13(4H, m), 4.65 (1H, dd, J=9.6, 2.4 Hz), 2.29 (3H, s), 1.38 (3H, d, J=6.6Hz).

¹³C NMR (75 MHz, CDCl₃): δ=169.35, 169.22, 162.73, 156.26, 150.41,135.79, 134.67, 133.77, 131.24, 128.35, 128.24, 128.08, 127.95, 125.78,123.51, 122.61, 71.22, 67.72, 67.26, 57.64, 20.98, 16.88.

The protected acetylsalicylic-L-threonine ester SPIB0010201 (14.50 g,28.68 mmole) was dissolved in ethanol (100 mL) and ethyl acetate (100mL) at room temperature and added to a Parr bottle that contained 10%palladium on carbon (3.0 g, 50% wet) under a nitrogen atmosphere. Thenitrogen atmosphere was replaced with hydrogen gas (20 psi). After 20hours of shaking, the palladium catalyst was removed by filtrationthrough celite. The remaining solids (palladium/celite and product) werewashed with water (600×4 mL) until the product was removed. The ethanoland water fractions were concentrated under reduced pressure at roomtemperature. The remaining solids were washed with water (20 mL) anddioxane (20 mL) for 48 hours. After filtration, the remaining whitesolid was dried at room temperature under high vacuum until the productweight was constant (16 hours). The experiment producedacetylsalicylic-L-threonine ester, SPIB00102 (4.40 g, 55% yield) as awhite solid.

¹H NMR (300 MHz, D₂O-DCl): δ=8.00 (1H, dd, J=7.8, 1.5 Hz), 7.74 (1H, dt,J=7.8, 1.5 Hz), 7.47 (1H, dt, J=7.8, 1.5 Hz), 7.27 (1H, dd, J=7.8, 1.5Hz), 5.76 (1H, dq, J=6.9, 3.0 Hz), 4.49 (1H, d, J=3.0 Hz), 2.39 (3H, s),1.55 (3H, d, J=6.9 Hz).

¹³C NMR (75 MHz, D₂O-DCl): δ=173.03, 168.84, 163.97, 149.56, 135.32,131.26, 126.85, 123.48, 121.49, 69.16, 56.36, 20.45, 15.86.

HPLC Analysis:

98.7% purity; rt=6.233 min; Luna C18 5 u column (sn 167917-13); 4.6×250mm; 254 nm; 35% MeOH/65% TFA (0.1%) pH=1.95; 35 C; 20 ul inj.; 1 ml/min;sample dissolved in mobile phase with 1 drop phosphoric acid.

CHN Analysis:

calc.: C, 55.51; H, 5.38; N, 4.98. found: C, 55.37; H, 5.40; N, 5.03.

Melting point: 153.5° C. (dec.)

2) SPIB00101 2-O-Acetylsalicylic acid (2S)-(+)-serine ester

A mixture of N-carbobenzyloxy-L-serine benzyl ester (Z-Ser-OBzl, 23.17g, 70.34 mmole) and pyridine (30 mL) in anhydrous dichloromethane (500mL) was cooled in an ice bath while under a nitrogen atmosphere.Acetylsalicyloyl chloride (21.07 g, 106.1 mmole) was added and themixture was allowed to warm to room temperature and stir over two days.After 48 hours, the mixture was poured into ice-cold 2N hydrochloricacid (400 mL). After mixing, the layers were separated and thedichloromethane fraction was first washed with water (500 mL), thensaturated sodium bicarbonate solution (500 mL), then water (500 mL),then brine (500 mL) and dried over sodium sulfate (25 g). Afterfiltration, concentration under reduced pressure, and drying under highvacuum, the remaining brown solid (47.19 g) was purified by flashchromatography on silica gel (200 g, 0.035-0.070 mm, 6 nm porediameter), eluting with hexanes/ethyl acetate (3:1). After concentrationof the product containing fractions under reduced pressure and dryingunder high vacuum until the weight was constant, the experiment producedthe protected acetylsalicylic-L-serine ester SPIB0010101 (32.97 g, 95%yield) as a white solid.

¹H NMR (300 MHz, CDCl₃): δ=7.74 (1H, d, J=7.8 Hz), 7.55 (1H, dt, J=7.8,1.5 Hz), 7.33-7.21 (11H, m), 7.08 (1H, d, J=7.5 Hz), 5.68 (1H, d, J=8.4Hz), 5.20 (2H, s), 5.12 (2H, s), 4.77 (1H, m), 4.66 (1H, dd, J=11.4, 3.3Hz), 4.57 (1H, dd, J=11.4, 3.3 Hz), 2.30 (3H, s).

¹³C NMR (75 MHz, CDCl₃): δ=169.45, 169.09, 163.68, 163.35, 155.57,150.77, 135.87, 134.75, 134.07, 131.44, 128.50, 128.43, 128.27, 128.14,128.04, 125.92, 123.71, 122.18, 67.83, 67.27, 64.63, 53.55, 21.03.

The protected acetylsalicylic-L-serine ester SPIB0010101 (21.0 g, 42.7mmole) was dissolved in ethanol (100 mL) and ethyl acetate (100 mL) atroom temperature and added to a Parr bottle that contained 10% palladiumon carbon (4.20 g, 50% wet) under a nitrogen atmosphere. The nitrogenatmosphere was replaced with hydrogen gas (20 psi). After 5 hoursadditional 10% palladium catalyst (4.26 g) was added and the hydrogenatmosphere was returned (20 psi). After an additional 20 hours ofshaking at room temperature, the palladium catalyst was removed byfiltration through celite. The remaining solids (palladium/celite andproduct) were washed with water (1500×2 mL) until the product wasremoved. The ethanol and water fractions were concentrated under reducedpressure at room temperature. The remaining solid (7.17 g) was dissolvedin DIUF water (4.3 L), filtered through celite to remove insolublematerial, and concentrated under high vacuum at room temperature. Thewhite solid was then washed with 1,4-dioxane (100 mL) and DIUF water (50mL) overnight. After 24 hours the solid was filtered and dried underhigh vacuum until the weight was constant (24 hours). The experimentproduced the acetylsalicylic-L-serine ester SPIB00101 (6.17 g, 54%yield) as a white solid.

¹H NMR (300 MHz, D₂O-DCl): δ=8.05 (1H, dd, J=7.8, 1.5 Hz), 7.75 (1H, dt,J=7.8, 1.5 Hz), 7.47 (1H, dt, J=7.8, 0.9 Hz), 7.27 (1H, dd, J=7.8, 0.9Hz), 4.87 (1H, dd, J=12.6, 4.2 Hz), 4.79 (1H, dd, J=12.6, 3.0 Hz), 4.62(1H, dd, J=4.2, 3.0 Hz), 2.39 (3H, s).

¹³C NMR (75 MHz, D₂O-DCl): δ=173.01, 168.58, 164.54, 149.72, 135.39,131.59, 126.87, 123.62, 121.15, 62.38, 52.05, 20.44.

HPLC Analysis:

98.1% purity; r.t.=5.839 min.; 65% TFA (0.1%)/35% methanol; 1 mL/min; 35C; Luna C18, 3 u column (SN 184225-37), 4.6×250 mm; 22 ul injection;DAD1B, Sig=240, 4 Ref=550, 100.

CHN Analysis:

calc.: C, 53.93; H, 4.90; N, 5.24. found: C, 54.02; H, 5.00; N, 5.23.

Melting point: 147.0° C. (dec.)

3) SPIB00103 2-O-Acetylsalicylic acid (2S,4R)-4-hydroxyproline ester

A mixture of N-carbobenzyloxy-L-hydroxyproline benzyl ester (Z-Ser-OBzl,21.5 g, 60.5 mmole) and pyridine (25 mL) in anhydrous dichloromethane(500 mL) was cooled in an ice bath while under a nitrogen atmosphere.Acetylsalicyloyl chloride (13.2 g, 66.6 mmole) was added and the mixturewas allowed to warm to room temperature and stir overnight. After 24hours, additional acetylsalicyloyl chloride (5.0 g, 25.2 mmole) wasadded and the mixture was allowed to stir overnight. After 48 hours, themixture was poured into ice-cold 1N hydrochloric acid (500 mL). Aftermixing, the layers were separated and the dichloromethane fraction waswashed with water (500 mL), then saturated sodium bicarbonate solution(500 mL), then water (500 mL), then brine (500 mL) and dried over sodiumsulfate (25 g). After filtration, concentration under reduced pressure,and drying under high vacuum, the remaining yellow oil (40.7 g) waspurified by flash chromatography on silica gel (460 g, 0.035-0.070 mm, 6nm pore diameter), eluting with heptane/ethyl acetate (3:1). Afterconcentration of the fractions containing product under reduced pressureand drying under high vacuum until the weight was constant, theexperiment produced the protected acetylsalicylic-L-hydroxyproline esterSPIB0010301 (21.31 g, 68% yield) as a colorless oil.

¹H NMR (300 MHz, CDCl₃): δ=7.92 (1H, d, J=7.8 Hz), 7.56 (1H, t, J=7.8Hz), 7.34-7.21 (10H, m), 7.09 (1H, d, J=7.8 Hz), 5.48 (1H, s), 5.21 (2H,m), 5.03 (2H, d, J=15 Hz), 4.57 (1H, m), 3.85 (2H, m), 2.53 (1H, m),2.28 (4H, m).

¹³C NMR (75 MHz, CDCl₃): δ=171.72, 171.49, 169.25, 163.47, 163.30,154.52, 153.93, 150.54, 136.05, 135.94, 135.21, 135.00, 134.17, 134.12,128.43, 128.32, 128.28, 128.20, 128.05, 127.98, 127.94, 127.79, 125.89,123.70, 122.46, 122.38, 73.24, 72.59, 67.33, 67.11, 66.97, 58.02, 57.69,52.47, 52.15, 36.74, 35.65, 20.90.

The protected acetylsalicylic-L-hydroxyproline ester SPIB0010301 (10.6g, 20.5 mmole) was dissolved in ethanol (75 mL) and ethyl acetate (75mL) at room temperature and added to a Parr bottle that contained 10%palladium on carbon (3.0 g, 50% wet) under a nitrogen atmosphere. Thenitrogen atmosphere was replaced with hydrogen gas (20 psi). After 17hours of shaking at room temperature, the reaction mixture was washedwith water (500 mL) for two hours. The organic layer (top) was removedvia pipette and the aqueous layer was filtered through celite. The waterfraction was concentrated under reduced pressure at room temperature.The remaining solid (6.71 g) was then washed with anhydrousdichloromethane (35 mL) overnight. After 24 hours the solid was filteredand dried under high vacuum until the weight was constant (24 hours).The experiment produced acetylsalicylic-L-hydroxyproline ester,SPIB00301 (2.87 g, 47.7% yield) as a white solid.

¹H NMR (300 MHz, D₂O-DCl): δ=8.09 (1H, d, J=7.5 Hz), 7.75 (1H, t, J=7.5Hz), 7.48 (1H, t, J=7.5 Hz), 7.28 (1H, d, J=7.5 Hz), 5.69 (1H, m), 4.76(1H, t, J=7.5 Hz), 3.86 (1H, dd, J=13.5, 3.9 Hz), 3.74 (1H, d, J=13.5Hz), 2.81 (1H, dd, J=15.0, 7.5 Hz), 2.60 (1H, m), 2.40 (3H, s).

¹³C NMR (75 MHz, D₂O-DCl): δ=173.13, 170.25, 164.31, 149.65, 135.36,131.54, 126.87, 123.54, 121.37, 73.86, 58.34, 50.95, 34.38, 20.48.

HPLC Analysis:

98.3% purity; r.t.=7.201 min.; 65% TFA (0.1%)/35% methanol; 1 mL/min; 35C; Luna C18, 3 u column (SN 184225-37), 4.6×250 mm; 22 ul injection;DAD1B, Sig=240, 4 Ref=550, 100.

CHN Analysis:

calc.: C, 57.34; H, 5.16; N, 4.78. found: C, 57.09; H, 5.23; N, 4.91.

Melting point: 162° C. (dec.)

Comparison of the L-Serine, L-Threonine, and L-Hydroxyproline Esters ofAcetylsalicylic Acid to Acetylsalicylic Acid with Respect to GastricMucosa Irritation

The present study was conducted to determine the relative potential ofthe new formulations of aspirin (L-serine, L-threonine, andL-Hydroxyproline esters of acetylsalicylic acid) to cause gastricmucosal irritation/lesions in fasted male albino rats. Aspirin served asa reference control.

Different new formulations of aspirin and aspirin were administered bygavage to fasted male albino rats (Wistar strain), using 0.5% (w/v)Carboxymethylcellulose (CMC) in Phosphate Buffer (pH, 2.6) solution asthe vehicle. The study was conducted at two dose levels viz. 100 mg and200 mg/kg body weight along with a vehicle control group. At each doselevel 5 animals were used. All the doses were expressed as aspirin molarequivalents. The doses used as well as the molar equivalents arepresented below.

TABLE 16 Formulation: Molar equivalent Formulation Molar equivalentL-serine ester of 1.483 units are equivalent acetylsalicylic acid to 1unit of aspirin L-Hydroxyproline ester 1.628 units are equivalent ofacetylsalicylic acid to 1 unit of aspirin L-threonine ester of 1.561units are equivalent acetylsalicylic acid to 1 unit of aspirin.

TABLE 17 Test Item: Group: Dose (mg per kg) [in terms of acetylsalicylicacid]: Equivalent weight of the Test item [mg] Dose (mg per kg) [interms of Equivalent acetylsalicylic weight of the Test Item Group acid]Test item [mg] Vehicle control Vehicle control 0.0 — Group L-serineester of Test Group 1 100.0 148.3 acetylsalicylic acid L-serine ester ofTest Group 1 100.0 148.3 acetylsalicylic Test Group 2 200.0 296.6 acidL-Hydroxyproline Test Group 1 100.0 162.8 ester of Test Group 2 200.0325.6 acetylsalicylic acid L-threonine, ester Test Group 1 100.0 156.1of acetylsalicylic Test Group 2 200.0 312.2 acid Reference control TestGroup 1 100.0 100.0 acetylsalicylic Test Group 2 200.0 200.0 acid

The rats were fasted for a period of 18 to 22 hours before dosing. Thetest item was administered as a single dose by gavage. Three hours afterdrug administration, the animals were killed humanely by CO₂ gasinhalation. The stomach was dissected out and observed for

-   -   the quantity of mucous exudate,    -   degree of hyperemia and thickening of stomach wall,    -   hemorrhagic spots (focal or diffuse), nature of hemorrhages        (petechial or ecchymotic) along with the size and    -   perforations

The observations on gastric mucosal irritation of animals of variousgroups are summarized below:

TABLE 18 Test Item: Group: Dose mg/kg (as acetylsalicylic acid):Observation Dose mg/kg (as acetylsalicylic Test Item Group acidObservation Vehicle control Vehicle None of the animals control Group0.0 showed any evidence of gastric mucosal irritation L-serine esterTest Group 1 100.0 None of the dosed of acetylsalicylic animals showedany acid evidence of gastric mucosal irritation Test Group 2 200.0 Noneof the dosed animals showed any evidence of gastric mucosal irritation.L-Hydroxyprolin Test Group 1 100.0 None of the dosed ester of animalsshowed any acetylsalicylic acid evidence of gastric mucosal irritation.Test Group 2 200.0 None of the dosed animals showed any evidence ofgastric mucosal irritation L-threonine, ester Test Group 1 100.0 None ofthe dosed of acetylsalicylic animals showed any acid evidence of gastricmucosal irritation Test Group 2 200.0 None of the dosed animals showedany evidence of gastric mucosal irritation Reference control Test Group1 100.0 None of the dosed (acetylsalicylic animals showed any acid)evidence of gastric mucosal irritation Test Group 2 200.0 All the 5animals dosed, showed evidence of gastric mucosal irritation.

In conclusion it was observed that none of the L-serine, L-threonine,and L-Hydroxyproline esters of acetylsalicylic acid induced any evidenceof irritation of gastric mucosa at the two doses tested viz., 100 and200 mg/kg body weight. In contrast, aspirin (acetylsalicylic acid)caused irritation of the gastric mucosal in all the fasted male albinorats at the dose level of 200 mg/kg. However at the dose level of 100mg/kg aspirin failed to cause any evidence of gastric mucosal irritationin the male rats. Further none of the animals of different test groupsshowed any clinical symptoms of toxicity during the observation periodof three hours.

Efficacy of L-Serine, L-Threonine, and L-Hydroxyproline Esters ofAcetylsalicylic Acid Compared to Acetylsalicylic Acid on Clotting Timein Rats Observations of Blood Clotting Time

The data on the mean clotting time (MCT) of the animals of low,intermediate and high dose groups of different formulations, vehiclecontrol and positive control groups estimated one hour after dosing werepresented below (Table 19):

TABLE 19 Summary of Mean Clotting Time (±S.D.) in Minutes-L-serine,L-threonine, and L-Hydroxyproline esters of acetylsalicylic acid andAspirin (Positive control): Low dose: Intermediate dose: High dose LowDose Intermediate Dose High Dose Vehicle control 4.9 ± 1.10 L-serineester of 5.7 ± 1.34 6.8 ± 1.48 6.9 ± 1.37 acetylsalicylic acidL-Hydroxyprolin ester 6.1 ± 1.10 5.7 ± 0.82 7.5 ± 1.18 ofacetylsalicylic acid L-threonine, ester of 5.2 ± 1.14 5.6 ± 0.84 7.4 ±0.97 acetylsalicylic acid Positive control 6.2 ± 1.40 8.1 ± 1.97 9.8 ±1.32 (acetylsalicylic acid)FIG. 3-6 depict the group mean data of animals regarding the doserelationship+mean clotting time in minutes for the L-series ester ofaspirin and for the control.

The statistical analysis showed a significant improvement at 5%significance level in the efficacy for the high dose and mid dose whencompared to the vehicle control group (FIG. 7).

FIG. 4 shows the group mean data of animals. It provides the doseresponse relationship to mean clotting time (MCT) in minutes withrespect to L-hydroxyproline ester of aspirin. The statistical analysisof FIG. 4 showed a significant improvement at 5% significance level inthe efficacy for the high dose and low dose when compared to the vehiclecontrol group (FIG. 6)FIG. 5 depicts the dose response relationship to mean clotting time(MCT) in minutes of L-threonine ester of acetylsalicylic acid. Thestatistical analysis showed a significant improvement at 5% significancelevel in the efficacy for the high dose when compared to the vehiclecontrol.FIG. 6 depicts the dose response relationship to mean clotting time foracetylsalicylic acid. The statistical analysis showed a significantimprovement at 5% significance level in the efficacy for theintermediate and high dose when compared to the vehicle control. Thedose response effect were statistically significant and clearly evident(FIG. 7).

Conclusion

The present study was conducted to evaluate the efficacy of newformulations of aspirin using blood clotting time as an index in albinorats. Aspirin served as positive control. The study was conducted atthree dose levels with the new formulations and positive control alongwith a vehicle control group.

Doses

The doses for the main study were selected based on the dose rangefinding experiments with acetylsalicylic acid. All the doses wereexpressed as aspirin molar equivalents. The doses used are presentedbelow.

TABLE 20 Test Item: Low Dose (mg/kg): Intermediate dose 9 mg/kg): Highdose (mg/kg) Low Dose Intermediate High Dose Test Item (mg/kg) Dose(mg/kg) (mg/kg) L-serine ester of 1.0 4.0 10.0 acetylsalicylic acidL-Hydroxyprolin ester 1.0 4.0 10.0 of acetylsalicylic acid L-threonine,ester of 1.0 4.0 10.0 acetylsalicylic acid Aspirin (Positive 1.0 4.010.0 control)

Efficacy (Blood Clotting Time)

The efficacy in terms of time required for the blood clotting time atdifferent dose levels—low, intermediate and high dose for differentformulations of various amino acid derivatives of aspirin andacetylsalicylic acid are presented below.

TABLE 21 Low dose: Intermediate dose: High dose Low Dose IntermediateDose High Dose Vehicle control 4.9 ± 1.10 L-serine ester of 5.7 ± 1.346.8 ± 1.48 6.9 ± 1.37 acetylsalicylic acid L-Hydroxyprolin ester 6.1 ±1.10 5.7 ± 0.82 7.5 ± 1.18 of acetylsalicylic acid L-threonine, ester of5.2 ± 1.14 5.6 ± 0.84 7.4 ± 0.97 acetylsalicylic acid Positive control6.2 ± 1.40 8.1 ± 1.97 9.8 ± 1.32

L-serine, L-threonine, and L-Hydroxyproline esters of acetylsalicylicacid are as effective as acetylsalicylic acid with respect to clottingtime observed after one hour after administration but are far superiorin terms of the absence of gastric irritation at all levels compared toacetylsalicylic acid.

Efficacy of L-Serine, L-Threonine, and L-Hydroxyproline Esters ofAcetylsalicylic Acid Compared to Acetylsalicylic Acid on Clotting Timein Rats Estimated Two Hours After Dosing

The present study was conducted to evaluate the efficacy of L-serine,L-threonine, and L-Hydroxyproline esters of acetylsalicylic acidcompared to acetylsalicylic acid using blood clotting time, estimated 2hours (±10 minutes) after dosing, as an index in albino rats. Aspirinserved as positive control. Male albino rats were exposed to aspirin andto 3 new formulations of amino acid derivatives of aspirin derivativesat one dose level of 20 mg/kg body weight. No vehicle control group wasused. The doses were expressed as aspirin molar equivalents. The dosesused for the main experiment for different formulations and positivecontrol was presented below.

TABLE 22 Test Item: Dose in terms of Acetylsalicylic acid 9 mg/kg) Dosein terms of Test Item Acetylsalicylic acid (mg/kg) L-serine ester ofacetylsalicylic acid 20.0 L-Hydroxyproline ester of 20.0 acetylsalicylicacid L-threonine ester of acetylsalicylic acid 20.0 Aspirin (Positivecontrol) 20.0

Efficacy (Blood Clotting Time)

The efficacy in terms of time required for the blood clotting time atthe dose level of 20 mg/kg body weight for different formulations ofamino acid derivatives of aspirin and aspirin (positive control) arepresented below:

Observations of Blood Clotting Time

The data on the mean clotting time (MCT) of the animals, estimated 2hours (±10 minutes) after dosing, at the dose level of 20 mg/kg bodyweight for the formulations of aspirin derivatives, vehicle control andpositive control are presented below

TABLE 23 Summary of Mean Clotting Time (±S.D.) in Minutes of L-serine,L-threonine, and L-Hydroxyproline esters of acetylsalicylic acidcompared to acetylsalicylic acid (Positive control) Dose (20 mg/kg)L-serine ester of acetylsalicylic acid 3.8 ± 0.92 L-Hydroxyproline esterof acetylsalicylic acid 4.2 ± 1.32 L-threonine ester of acetylsalicylicacid 5.3 ± 1.06 Positive control (acetylsalicylic acid) 5.4 ± 1.17L-serine, L-threonine, and L-Hydroxyproline esters of acetylsalicylicacid were found to be effective on clotting time.

In conclusion, it was observed that based on the time required for theblood to clot (clotting time), when estimated 2 hours after dosing, theamino acid derivatives were efficacous. However, the L-threonine esterof acetylsalicyclic acid was found to have relatively better efficacythan the other two formulations.

As shown by FIG. 7 the statistical analysis showed that L-threonine, andL-Hydroxyproline esters of acetylsalicylic acid are at least aseffective as acetylsalicylic acid. There is no significant difference at5% significance level for L-Hydroxyproline ester of acetylsalicylic acidand L-threonine ester of acetylsalicylic with respect to positivecontrol for the mean blood clotting time observed after two hours.However, combined with the gastric irritation potential, the L-serine,L-threonine, and L-Hydroxyproline esters of acetylsalicylic acid are farsuperior.

The following additional data were obtained, when the same drugs werecompared in rats on a different date with different time intervals anddoses:

TABLE 24 Clotting Time (min) Dose ASA-Ser ASA-Hyp ASA-Thr ASA Vehicle 10mg/kg 1 hr 6.9 7.5 7.4 9.8 4.9 10 mg/kg 24 hr 4.4 3.3 3.6 4.4 2.7 20mg/kg 2 hr 3.8 4.2 5.3 5.4 2.7 *ASA is acetylsalicyclic acid Two of thebetter esters of ASA at various conditions are as follows: 10 mg/kg 1 hrASA-Hyp and ASA-Thr 10 mg/kg 24 hr ASA-Serine and ASA-Thr 20 mg/kg 2 hrASA-Hyp and ASA-Thr

When compared against the various amino acid esters, L-Threonine Esterof Aspirin seems to be the most preferred as it showed consistently abetter response in the rat blood clotting time.

Based upon consistently improved toxicity profile, efficacy and bettertherapeutic index, Acetylsalicylic Acid-L-Threonine Ester was advancedto GMP synthesis.

Results of the 28-Day Chronic Dosing in Rodents, Comparative Toxicology:

The purpose of this study is to establish the toxicity ofAcetylsalicylic Acid-L-Threonine Ester in relation to Aspirin (Make:Sigma, Batch number 090K0884) which served as a reference drug byconducting a 28-day repeated dose oral toxicity test in male and femalealbino rats.

Aspirin and Acetylsalicylic Acid-L-Threonine Ester were administered toalbino rats (Wistar strain), by oral gavage daily for a period of 28days, using 0.5% Carboxymethylcellulose (CMC) in phosphate buffersolution (pH, 2.6) as vehicle. The study was conducted at one dose levelonly along with a vehicle control group as per the recommendation of theSponsor. The test doses are expressed as Aspirin molar equivalents.Acetylsalicylic Acid-L-Threonine Ester was compared against Aspirin andVehicle at 100 mg/kg dose administered to rats for 28 days.

The salient features of the study are as follows, where ASA-T representsAcetylsalicylic Acid-L-Threonine Ester:

-   -   1. All the animals of vehicle control group and the test group        (Acetylsalicylic Acid-L-Threonine Ester) and reference control        group (Aspirin) survived through the dosing period of 28 days.    -   2. None of the animals of the vehicle control group, test group        (Acetylsalicylic Acid-L-Threonine Ester), and reference control        group (Aspirin) exhibited any clinical symptoms of toxicity        through out the dosing period.    -   3. The Changes in the Body weight is tabulated hereinbelow.

TABLE 25 Body Weight Comparison Significance[P < 0.05] Gain ASA-T vsAspirin vs Vehicle Normal (Male) Gain ASA-T and Aspirin vs VehicleDecrease (Female)The percentage decrease were 29% and 21% for AcetylsalicylicAcid-L-Threonine Ester and Aspirin

-   -   4. Food intake of the animals of both the sexes of test group        (Acetylsalicylic Acid-L-Threonine Ester) and reference control        group (Aspirin) was found to be normal and comparable to the        animals of vehicle control group.    -   5. Results of hematological analysis of the animals of different        groups are shown in Table 26 below:

TABLE 26 Hematological Comparison Significance[P < 0.05] All BloodParameters ASA-T vs Aspirin vs Vehicle None Platelet Count ASA-T vsAspirin Increase (Male)

-   -   6. Results of clinical chemistry analysis of the animals of        different groups are summarized below in Table 27:

TABLE 27 Clinical Chemistry Comparison Significance[P < 0.05] AlkalinePhosphatase AST-T Ester vs Vehicle Increase (Female) Total Protein ASA-TEster ® vs Vehicle Increase (Female) Creatinine ASA-T Ester ® vs VehicleDecrease (Male) Cholesterol ASA-T Ester ® vs Vehicle Increase (Male)Alkaline Phosphatase Aspirin vs Vehicle Increase (Female) Sodium Aspirinvs Vehicle Increase (Female) Blood Urea Aspirin vs Vehicle Decrease(Female) SGPT and Cholesterol Aspirin vs Vehicle Increase (Male) AllClinical Chemistry ASA-T Ester ® vs Aspirin None(but two below) BloodGlucose ASA-T Ester ® vs Aspirin Decrease (Female) Creatinine ASA-TEster ® vs Aspirin Decrease (Male)

-   -   7. Necropsy of the surviving animals at the end of the study        (terminal necropsy) of vehicle control and different treatment        groups did not reveal any gross pathological changes in any of        the vital organs. Further there is no evidence of gastric        mucosal irritation in the animals of vehicle control, test group        (ASA-T Ester) and reference control group (Aspirin).    -   8. The data on absolute (Abs) and relative (Rel) organ weights        of liver, kidney, adrenals, heart, spleen and testes showed the        following changes in the organ weights are depicted in Table 28:

TABLE 28 Rel/Abs Organ Wts Comparison Significance[P < 0.05] Adrenals(Abs) ASA-T Ester ® vs Vehicle Decrease (Male) Kidney (Abs) Aspirin vsVehicle Decrease (Male) Spleen (Abs) Aspirin vs Vehicle Decrease(Female) Kidney (Rel) Aspirin vs Vehicle Increase (Male) Spleen (Rel)Aspirin vs Vehicle Increase (Female) Kidney (Abs) ASA-T Ester vs AspirinDecrease (Male) Spleen (Abs) ASA-T Ester vs Aspirin Decrease (Male)Kidney (Rel) ASA-T Ester vs Aspirin Increase (Male)

-   -   9. Histological sections of the following organs viz. brain,        stomach, small intestines, large intestine, liver, kidney,        adrenal, spleen, heart, lungs and gonads of male and female        animals treated with Acetylsalicylic Acid-L-Threonine Ester or        reference drug (Aspirin) groups did not show any        histopathological changes and were found to be normal and        comparable to that of animals of vehicle control group. However        few animals treated with reference drug (Aspirin) showed mild        fatty changes in the cardiac muscle fibers of heart and mild        catarrhal changes of gastric mucosa.

Human Clinical Trials:

Several blood clotting time studies were performed on a limited numberof human volunteers with Acetylsalicylic Acid-L-Threonine Ester. Resultsare shown in FIGS. 8-11. Attention is directed to FIG. 8 which comparesthe average clotting time after 325 mg and 81 mg Acetylsalicyclic AcidL-Threonine Ester and Bayer Aspirin is administered to human volunteers.The first set of columns in FIG. 8 labeled “Normal” is the averageclotting time observed prior to the administration of the Aspirin or itsthreonine ester derivative thereof.

As clearly shown by the data in FIG. 8, Acetylsalicylic Acid-L-ThreonineEster at low dose of 80 mg was as effective as 325 mg Bayer Aspirin(with baseline correction), and obviously more effective than Bayer 81mg Aspirin.

The data are tabulated in Table 29. The data on which the graphs of FIG.8 are based are summarized in Table 29 below:

TABLE 29 AVERAGE CLOTTING TIME (MIN) 325 mg 325 mg 81 mg 81 mg 81 mgASA-T ASA ASA-T ASA ASA-T ESTER Bayer ESTER Bayer ESTER Volun- Volun-Volun- Volun- Volun- teer 1 teer 2 teer 1 teer 2 teer 3 Normal 4.5 4.5 44 4 With ASA-T ESTER 6.2 6.0 5.2 NA 5.1 With ASA (Bayer) 5.7 5.7 NA 4.5NA

FIG. 10.0 depicts the percentage increase in clotting time ofAcetylsalicylic Acid-L-Threonine Ester relative to Aspirin (Bayer) at 81mg dose based on 5-day average increase. This plot was derived from thedata in Table 29. The two Acetylsalicylic Acid-L-Threonine Ester blocksshown above correspond to two separate volunteers who took the test drugover a period of 5 days. The third volunteer took Bayer Aspirin for 5days. As per FIG. 2, increase in clotting time occurred on the veryfirst day of drug intake, and remained higher than Bayer ASA during thesubsequent administrations.

Pharmacokinetic Results in 4 volunteers who took AcetylsalicylicAcid-L-Threonine Ester versus two volunteers who took Bayer Aspirin at325 mg dose are shown in FIG. 11.

FIG. 11 is a plot of the concentration of Aspirin versus time in 4volunteers who took Acetylsalicylic Acid-L-Threonine Ester and 2volunteers who took Bayer Aspirin. No Aspirin was found in blood forvolunteers who took Acetylsalicylic Acid-L-Threonine Ester. The twovolunteers who took Bayer Aspirin showed Plasma levels of Aspirin.

This is shown in FIG. 12 which depicts the concentration of salicyclicAcid in human plasma with respect to four volunteers who tookAcetylsalicyclic Acid-L-Threonine Ester and two volunteers, who tookAspirin.

Rapid influx and efflux of Salicylic Acid from Aspirin administrationwas seen in the two volunteers who took Aspirin (FIG. 12). There wasprolonged and sustained formation and disappearance of Salicylic acid involunteers who took Acetylsalicylic Acid-L-Threonine Ester (FIG. 7).Without wishing to be bound, this phenomenon is believed to beattributed to the high likelihood of Acetylsalicylic Acid-L-ThreonineEster exhibiting sustained, specific and irreversible acetylation of theplatelets in the portal circulation. Since there was no aspirin found inthe systemic circulation after oral dosing of AcetylsalicylicAcid-L-Threonine Ester, it is likely that site specific action ofacetylation of the platelets has been achieved, and the unwanted effectof aspirin in the endothelial system has been avoided. Thus it isevident from the human pharmacokinetic studies, clinical trials, and ratgastric mucosa irritation results, Acetylsalicylic Acid-L-ThreonineEster is a superior anti-platelet drug that does not have the toxicityof Aspirin and which exhibits a much better Therapeutic Index.

Therefore, Acetylsalicylic Acid-L-Threonine Ester is a Superior Antiplatelet drug than acetylsalicyclic acid, for several reasons:

-   -   1. It does not produce any gastric irritation;    -   2. It does not inhibit prostaglandin synthesis;    -   3. It does not have any COX-1 or COX-2 activity on the        endothelial or vascular tissues, as no ASA from Acetylsalicylic        Acid-L-Threonine Ester reaches systemic circulation.    -   4. Thus, none of the side effects of Aspirin is observed with        Acetylsalicylic Acid-L-Threonine Ester.

There are a number of screening tests to determine the utility of thederivatives created according to the disclosed methods. These includeboth in vitro and in vivo screening methods.

The in vitro methods include acid/base hydrolysis of the derivatives,hydrolysis in pig pancreas hydrolysis in rat intestinal fluid,hydrolysis in human gastric fluid, hydrolysis in human intestinal fluid,and hydrolysis in human blood plasma. These assays are described inSimmons, D M, Chandran, V R and Portmann, G A, Danazol Amino AcidDerivatives: In Vitro and In Situ Biopharmaceutical Evaluation, DrugDevelopment and Industrial Pharmacy, Vol 21, Issue 6, Page 687, 1995,the contents of all of which are incorporated by reference.

The NSAID amino acid derivatives of the present invention are effectivein treating diseases or conditions in which NSAIDs normally are used.These amino acid derivatives disclosed herein enhance the therapeuticbenefits of the NSAIDs by reducing or eliminating biopharmaceutical andpharmacokenetic barriers associated with each of them. However it shouldbe noted that these amino acid derivatives themselves will havesufficient activity without releasing any active drug in the mammals.Since the amino derivatives are more soluble in water than Ibuprofen orother NSAIDs, they do not need to be associated with a carrier vehicle,such as alcohol or castor oil which may be toxic or produce unwantedside reactions. Moreover, oral formulations containing the NSAIDderivatives are absorbed into the blood and are quite effective.

Thus, the amino acid derivatives of the present invention, discussedhereinabove enhance the therapeutic benefits by removingbiopharmaceutical and pharmacokenetic barriers of existing drugs.

Furthermore, these amino acid derivatives are easily synthesized in highyields using reagents which are readily and commercially available.

IV. Proline Derivative of Acetaminophen Overview:

The procedure for the synthesis of the L-proline ester of acetaminophenis outlined in Synthetic Sequence section. The synthesis is exemplary.The complete procedure and analytical data is given in the ExperimentalSection. Acetaminophen (10 g) was coupled with Boc-L-proline with EDC inthe presence of a catalytic amount of DMAP. Once the reaction wascomplete (3 hours at room temperature), the solution was washed withwater. After drying over sodium sulfate, filtration, and concentrationthe crude protected amino acid ester of acetaminophen was purified byflash chromatography on silica gel. The procedure generated theprotected L-proline ester of acetaminophen in 72%. The protecting groupwas removed by dissolving the ester in dichloromethane and passinghydrogen chloride through the solution at room temperature. Afterfiltration, the final salt was stirred in tetrahydrofuran until pure.The yield for the deprotection step was 91.4% after filtration anddrying under high vacuum at 90° C. for 4 hours.

Synthetic Sequence:

Synthesis of the L-proline ester of acetaminophen a) EDC, DMAP, CH₂Cl₂;b) HCl (g), CH₂Cl₂ Experimental Section:

The synthesis of SPI0014 was conducted in one batch. Reagents mentionedin the experimental section were purchased at the highest obtainablepurity from Lancaster, Sigma-Aldrich, or Acros, except for solvents,which were purchased from either Fisher Scientific or Mallinkrodt.

SPI0014 Pyrrolidine-2(S)-carboxylic acid 4-acetylamino-phenyl ester,hydrochloride

A mixture of Boc-L-proline (14.39 g, 68.80 mmole), acetaminophen (10.02g, 66.28 mmole), EDC (12.9 g, 67.29 mmole) and DMAP (1.10 g, 9.0 mmole)in anhydrous dichloromethane (100 mL) was stirred for 3 hours at roomtemperature under an argon atmosphere. After 3 hours, water (120 mL) wasadded. After mixing for 5 minutes, the layers were separated and thedichloromethane fraction was washed with water (120 mL) and dried oversodium sulfate (5 g). After filtration, concentration under reducedpressure, and drying under high vacuum, the remaining oil (24.10 g) waspurified by flash chromatography on silica gel (100 g, 0.035-0.070 mm, 6nm pore diameter), eluting with hexanes/ethyl acetate (1:2). Afterconcentration of the product containing fractions under reduced pressureand drying at high vacuum until the weight was constant, the experimentproduced the protected acetaminophen-L-proline ester SPI001401 (16.71 g,72.3% yield) as a white solid (foam).

¹H NMR (300 MHz, CDCl₃): δ=8.83 (½H, s), 8.70 (½H, s), 7.58 (½H, d,J=7.5 Hz), 7.46 (½H, d, J=7.5 Hz), 6.96 (2H, m), 4.47 (1H, m), 3.59-3.45(2H, m), 2.36 (1 H, m), 2.17-1.90 (6H, m), 1.46 (9H, m).

¹³C NMR (75 MHz, CDCl₃): δ=171.91, 171.75, 169.02, 154.44, 153.78,146.36, 146.21, 121.44, 121.23, 120.82, 80.41, 80.17, 59.16, 46.78,46.55, 31.06, 30.11, 28.50, 24.57, 24.28, 23.78.

The protected acetaminophen-L-proline ester SPI001401 (16.60 g, 47.64mmole) was dissolved in dichloromethane (400 mL) and hydrogen chloridegas was passed through the solution for 2 hours at room temperature. Theremaining solids were allowed to settle (for 1 hour). Thedichloromethane was carefully decanted away from the white precipitate.Tetrahydrofuran (200 mL) was added to the precipitate and the mixturestirred for 2 hours under an argon atmosphere. After filtration, theremaining white solid was dried under high vacuum at 90° C. until theproduct weight was constant (4 hours). The experiment producedacetaminophen-L-proline ester, hydrochloride SPI0014 (12.4 g, 91.4%yield) as a white solid.

¹H NMR (300 MHz, CDCL₃-DMSO): δ=10.41 (1H, br s), 10.26 (1H, s), 9.55(1H, br s), 7.70 (2H, d, J=9Hz), 7.12 (2H, d, J=9Hz), 4.66 (t, 1H, J=8.4Hz), 3.33 (2H, m), 2.43 (1H, m), 2.28 (1H, m), 2.08 (s, 3H), 2.04 (2H,m).

¹³C NMR (75 MHz, CDCL₃-DMSO): δ=168.08, 167.25, 144.55, 137.40, 121.12,119.64, 58.53, 45.33, 27.74, 23.86, 23.08.

HPLC Analysis:

99.45% purity; rt=5.733 min; Luna C18 5 u column (sn 167917-13); 4.6×250mm; 254 nm; 15% MeOH/85% hexane sulfonate buffer (110 mMol, pH=6); 35 C;20 ul inj.; 1 ml/min; 5 mg/mL sample size.

CHN Analysis:

calc.: C, 54.84; H, 6.02; N, 9.84. found: C, 54.66; H, 5.98; N, 9.65.

Melting point: 221-222° C.

V. Amino Acid Derivative of Cyclosporine A

The macrocyclic immunosuppresants comprise a class of structurallydistinctive, cyclic, poly, N-methylated undecaptides, and similarsemi-synthetic macrolide structures commonly possessing pharmacological,in particular immunosuppressive, anti-inflammatory and/or anti-parasiticactivity. The first of the cyclosporine to be isolated was the naturallyoccurring fungal metabolite Ciclosporin or Cyclosporine also known ascyclosporine A, which has the formula:

wherein MeBmt represents N-methyl-(4R)-4-but-2E-en-1-yl-4-methyl-(L)threonyl residue of the formula

in which -x-y- is CH═CH—(trans). Other similar products include,sirolimus (b), tacrolimus (c), and pimecrolimus (d), having thefollowing structures:

The class comprised by the cyclosporines is thus now very large indeedand includes, for example, [Thr]²-, [Val]²-, [Nva]²- and[Nva]²-[Nva]⁵-Ciclosporin (also known as cyclosporines C, D, G and Mrespectively), [Dihydrop-MeBmt]¹-[Val]²-ciclosporin (also known asdihydro-cyclosporine D), [(D)Ser]8-Ciclosporin, [MeIle]¹¹-Ciclosporin,[(D)MeVal]¹¹-Ciclosporin (also known as cyclosporine H),[MeAla]⁶-Ciclosporin, [(D)Pro]3-Ciclosporin and so on.

In accordance with conventional nomenclature for cyclosporines, theseare defined throughout the present specification and claims by referenceto the structure of cyclosporine (i.e., Cyclosporine A). This is done byfirst indicating the amino acid residues present which differ from thosepresent in cyclosporine (e.g., “[(D)Pro]³” to indicate that thecyclosporine in question has a -(D)Pro- rather than -Sar- residue at the3-position) and then applying the term Cyclosporine to characterizeremaining residues which are identical to those present in CyclosporineA.

As used herein, the term “cyclosporines” refers to the various types ofcyclosporines, in which x-y in the MeBmt residue has a cis or transCH═CH or in which x-y therein is also included in those derivatives inwhich one or more of those amino acids in positions 2-11 of CyclosporineA is replaced by a different amino acid. It is preferred, however, thatnot more than two of the amino acids are replaced in the formula ofcyclosporine A and more preferentially not more than one of the aminoacids is replaced by an amino acid.

In addition, amino acid residues referred to by abbreviation, e.g.,-Ala-, -MeVal- and -αAbu-, are, in accordance with conventionalpractice, to be understood as having the (L)-configuration unlessotherwise indicated, e.g. as in the case of “-(D)Ala-”. Residueabbreviations preceded by “Me” as in the case of “-MeLeu-”, representα-N-methylated residues. Individual residues of the cyclosporinemolecule are numbered, as in the art, clockwise and starting with theresidue -MeBmt-, dihydro-MeBmt- etc. . . . in position 1. The samenumerical sequence is employed throughout the present specification andclaims.

Because of their unique pharmaceutical potential, the macrocyclicimmunosuppressants have attracted considerable attention in the press.The term “macrocyclic immuno-suppressants” includes various natural andsemi-synthetic derivatives of cyclosporine, and other macrolides such assirolimus, tacrolimus and pimecrolimus. The primary area of clinicalinvestigation for the above drugs has been as immunosuppressive agents,in particular in relation to its application to recipients of organtransplants, e.g., heart, lung, combined heart-lung, liver, kidney,pancreatic, bone-marrow, skin and corneal transplants, and in particularallogenic organ transplants. These drugs are also used in the treatmentof psoriasis, atompic dermatitis, rheumatoid arthritis and nephriticsyndrome.

Macrocyclic immunosuppressants are also useful for treating variousautoimmune diseases and inflammatory conditions and especiallyinflammatory conditions with an aetiology, including an autoimmunecomponent, such as arthritis (for example, rheumatoid arthritis,arthritis chronica progredient and arthritis deformons) and rheumaticdiseases. Specific autoimmune diseases for which cyclosporine therapyhas been proposed or applied include, autoimmune hematological disorder(including, e.g., hemolytic anemia, aplastic anemia, pure red cellanemia, and idiopathic thrombocytopaenia), systemic lupus erythematosus,polychondritis, sclerodoma, Wegener granulamatosis, dermatomyositis,chronic active hepatitis, myasthenia gravis, psoriasis, Steven-Johnsonsyndrome, idiopathic sprue, autoimmune inflammatory bowel disease,including, e.g., ulcerative colitis and Crohn's disease), endocrineopthalmopathy Graves disease, sarcoidosis, multiple sclerosis, primarybilliary cirrhosis, juvenile diabetes (diabetes mellitus type I), uvetis(anterior and posterior), keratoconjunctivitis sicca and vernalkeratoconjunctivitis, interstial lung fibrosis, psoriatic arthritis,atopic dermatitis and glomerulonephritis (with and without nephroticsyndrome, e.g., including idiopathic nephritic syndrome or minimalchange nephropathy).

Furthermore, macrocyclic immunosuppressants also have applicability asan anti-parasitic, in particular anti-protozoal agent, and are suggestedto be useful for treating malaria, coccidiomycosis and schistomsomiasis.More recently, they have been taught to be useful as agents forreversing or abrogating anti-neoplastic agent resistance contumors, andthe like.

Despite the very major contribution which macrocyclic immunosuppressantshave made, difficulties have been encountered in providing moreeffective and convenient means of administration (e.g., galenicformulations, for example, oral dosage form, which are both convenientand for the patient as well as providing appropriate bioavailability andallowing dosaging at an appropriate and controlled dosage rate) as wellas the reported occurrence of undesirable side reactions; in particularnephrotoxic reactions have been obvious serious impediments to its wideruse or application.

Moreover, the above mentioned macrocyclic immunosuppressants arecharacteristically highly hydrophobic and readily precipitate in thepresence of even very minor amounts of water, e.g., on contact with thebody (e.g., stomach fluids). It is accordingly extremely difficult toprovide e.g., oral formulations, which are acceptable to the patient interms of form and taste, which are stable on storage and which can beadministered on a regular basis to provide suitable and controllingpatient dosaging.

Proposed liquid formulations, e.g., for oral administration ofmacrocyclic immunosuppressants, have heretofore been based primarily onthe use of ethanol and oils or similar excipients as carrier media.Thus, the commercially available macrocyclic immunosupressantdrink-solution employs ethanol and olive oil or corn-oil as carriermedium in conjunction with solvent systems comprising e.g., ethanol andLABRIFIL and equivalent excipients as carrier media. Thus, thecommercially available macrocyclic immunosupressant drink solutionemploys ethanol and olive oil or corn-oil as carrier medium inconjunctions with a Labrifil as a surfactant. See e.g., U.S. Pat. No.4,388,307. Use of the drink solution and similar composition as proposedin the art is, however, accompanied by a variety of difficulties.

Further, the palatability of the known oil based system has provedproblematic. The taste of the known drink-solution is, in particular,unpleasant. Admixture with an appropriate flavored drink, for example,chocolate drink preparation, at high dilution immediately prior toingestion has generally been practiced in order to make regular therapyat all acceptable. Adoption of oil based systems has also required theuse of high ethanol concentrations to itself inherently undesirable, inparticular where administration to children is forseen. In addition,evaporation of the ethanol, e.g., from capsules (adopted in large part,to meet problems of palatability, as discussed or other forms (e.g.,when opened)) results in the development of a macrocyclicimmunosupressant precipitate. When such compositions are presented in,for example, soft gelatin encapsulated form an additional problemarises. This particular difficulty necessitates packaging of theencapsulated product in an air-tight component, for example, anair-tight blister or aluminum-foil blister package. This in turn rendersthe product both bulky and more expensive to produce. The storagecharacteristics of the aforesaid formulations are, in addition, far fromideal.

Bioavailability levels achieved using existing oral macrocyclicimmunosupressant dosage system are also low and exhibit wide variationbetween individuals, individual patient types and even for singleindividuals at different times during the course of therapy. Reports inthe literature indicate that currently available therapy employing thecommercially available macrocyclic immunosupressant drink solutionprovides an average absolute bioavailability of approximately 30% only,with the marked variation between individual groups, e.g., between liver(relatively low bioavailability) and bone-marrow (relatively highbioavailability) transplant recipients. Reported variation inbioavailability between subjects has varied from one or a few percentfor some patients, to as much as 90% or more for others. And as alreadynoted, marked change in bioavailability for individuals with time isfrequently observed. Thus, there is a need for a more uniform and highbioavailability of macrocyclic immunosupressant in patients.

Use of such dosage forms is also characterized by extreme variation inrequired patient dosaging. To achieve effective immunosuppressivetherapy, blood or blood serum levels compounds of the cyclosporin haveto be maintained within a specified range. This required range can inturn, vary, depending on the particular condition being treated, e.g.,whether therapy is to prevent transplant rejection or for the control ofan autoimmune disease, or condition and on whether or not alternativeimmunosuppressive therapy is employed concomitantly with any of theimmunosuppressants of the formula described herein. Because of the widevariations in bioavailability levels achieved with conventional dosageforms, daily dosages needed to achieve required blood serum levels willalso vary considerably from individual to individual and even for asingle individual. For this reason it is necessary to monitorblood/blood-serum levels of patients receiving macrocyclicimmunosuppressant therapy at regular and frequent intervals. Monitoringof blood/blood-serum levels, which is generally performed by RIA orequivalent immunoassay technique, e.g. employing monoclonal antibodybased technology, has to be carried out on a regular basis. This isinevitably time consuming and inconvenient and adds substantially to theoverall cost of therapy.

It is also the case that blood/blood serum macrocyclic immunosuppressantlevels achieved using available dosage systems exhibit extreme variationbetween peak and trough levels. That is, for each patient, effectivemacrocyclic immunosuppressant levels in the blood vary widely betweenadministrations of individual dosages.

There is also a need for providing macrocyclic immunosuppressant in awater soluble form for injection. It is well known that Cremephore Lused in a current formulations of macrocyclic immunosuppressants is apolyoxyethylated derivative of castor oil and is a toxic vehicle. Therehave been a number of incidences of anaphylaxis due to the castor oilcomponent. At present there is no formulation that would allow themacrocyclic immunosuppressants to be in aqueous solution at theconcentrations needed due to poor water solubility of the drug.

Beyond all these very evident practical difficulties lies the occurrenceof undesirable side reactions already alluded to, observed employingavailable oral dosage forms.

Several proposals to meet these various problems have been suggested inthe art, including both solid and liquid oral dosage forms. Anoverriding difficulty which has, however, remained is the inherentinsolubility of the macrocyclic immunosuppressants in aqueous media,hence preventing the use of a dosage form which can contain macrocyclicimmunosuppressants in sufficiently high concentration to permitconvenient use and yet meet the required criteria in terms ofbioavailability, e.g. enabling effective resorption from the stomach orgut lumen and achievement of consistent and appropriately highblood/blood-serum levels.

The particular difficulties encountered in relation to oral dosagingwith macrocyclic immunosuppressants have inevitably led to restrictionsin the use of macrocyclic immunosuppressant therapy for the treatment ofboth relatively less severe or endangering disease conditions. Aparticular area of difficulty in this respect has been the adoption ofmacrocyclic immunosuppressant therapy in the treatment of autoimmunediseases and other conditions affecting the skin, for example, for thetreatment of atopic dermatitis and psoriasis and, as also widelyproposed in the art, for hair growth stimulation, e.g. in the treatmentof alopecia due to ageing or disease.

Thus while oral macrocyclic immunosuppressant therapy has shown that thedrug is of considerable potential benefit to patients suffering e.g.from psoriasis, the risk of side-reaction following oral therapy hasprevented common use. Various proposals have been made in the art forapplication of macrocyclic immunosuppressants, e.g. cyclosporine, intopical form and a number of topical delivery systems have beendescribed. Attempts at topical application have however failed toprovide any demonstrably effective therapy.

However, the present invention overcomes the problems describedhereinabove. More specifically, an embodiment of the present inventionis an amino acid derivative of macrocyclic immunosuppressant whichsignificantly enhances its solubility in aqueous solutions, therebyavoiding the need to utilize a carrier, such as ethanol or castor oilwhen administered as a solution. Moreover, the amino acid derivatives ofmacrocyclic immunosuppressants, in accordance with the presentinvention, do not exhibit the side effects of the prior artformulations. Further, the inventor has found that the macrocyclicimmunosuppressant amino acid derivatives of the present inventionenhance their absorption when administered in the form of the amino acidderivative to a patient, thereby enhancing significantly theirbioavailability and efficacy.

Accordingly, in one aspect, the present invention is directed to anamino acid derivative of macrocyclic immunosuppressants. The amino acidderivative consists of an amino acid esterified to the free hydroxygroup present on the macrocyclic immunosuppressants; e.g., on the sidechain of cyclosporine, sirolimus, tacrolimus and either one of thehydroxyl groups of the pimecrolimus molecule.

For example, an aspect of the present invention is directed to, thecompounds of the formulas

or pharmaceutically acceptable salts thereof;wherein CYCLO represents the residues at positions 2-11 of thecyclosporine molecule;x-y is CH═CH or CH₂CH₂ and AA is an amino acid or a dipeptide of theformula GLY-AA. In the latter case, GLY is glycine and AA is any α-aminoacid but preferably the L-α-amino acid and more preferably the naturallyoccurring amino acids, especially in the L-form. In the dipeptidestructure, an AA is attached to the drug via OH group using glycine asthe spacer. Glycine is esterified to cyclosporine and then glycine isbonded to any AA via amide linkage using amino group of glycine andcarboxylic acid group of AA.

The present invention is also directed to a pharmaceutical compositioncomprising a therapeutically effective amount of the compounds of theFormulae a-d above and a pharmaceutical carrier therefor.

In another embodiment, the present invention is directed to a method oftreating a patient in need of macrocyclic immunosuppressant therapy,which method comprises administering to said patient an effective amountof the compounds of Formulae a-d.

In a further embodiment, the present invention is directed to a methodof enhancing the solubility of a macrocyclic immunosuppressant in anaqueous solution comprising reacting said immunosuspressant having ahydroxy group thereof, e.g., hydroxy functionality in the MeBmt moietyat position 1 of the cyclosporine molecule as well as the specifiedhydroxyl functions in formulas b-d, with an amino acid or acylatingderivative thereof under ester forming conditions, and isolating theproduct or by using a simple amino acid or a dipeptide structure oracylating derivative wherein the AA is attached to drug using glycine asthe spacer and isolating and isolating the product thereof.

In a still further embodiment, the present invention is directed to amethod of enhancing the bioavailability of a macrocyclicimmunosuppressant when administered to a patient which comprisesreacting the immunosuppressant having a hydroxy group thereon, e.g., thehydroxy functionality in the MeBmt moiety in position 1 of thecyclosporine molecule, with an amino acid or acylating derivative underester forming conditions and as well as the specified hydroxyl functionsin formulas b-d with an amino acid or acylating derivative thereof underester forming conditions or by using a simple amino acid or a dipeptidestructure wherein the AA is attached to the drug using glycine as thespacer and isolating the product thereof and administering said productto the patient.

Overview:

The procedure for the synthesis of the N-(L-proline)-glycine andN-(L-lysine)-glycine esters of Cyclosporine A is outlined in SyntheticSequence section. These examples are exemplary of the synthetic schemeusing amino acids. The complete procedure and analytical data is givenin the Experimental Section. Cyclosporine A (15 g) was coupled withchloroacetic anhydride (4 equivalent) in anhydrous pyridine. Theexperiment produced the chloroacetate ester of Cyclosporine A(SPI001201, 14 g, 88% yield) in good yield. The chloroacetate ester(10.1 g) was then treated with sodium azide in DMF to generate theazidoacetate ester of Cyclosporine A (SPI001202, 9.9 g, 97% yield). Theazidoacetate (9.8 g) was then reduced with tin chloride (9 g) to preparethe glycine ester of Cyclosporine A (8.54 g, 89% yield). The glycineester of Cyclosporine A (SPI001203) was then coupled with a two-foldexcess of either boc-L-proline or Boc-L-lysine using EDC as the couplingagent. After purification by column chromatography, the boc protectinggroups were removed from the dipeptide esters of Cyclosporine A at lowtemperature (5° C.) by treatment with 2M hydrochloric acid in diethylether. The L-lysine-glycine ester salt of Cyclosporine A did not requireadditional purification and was dried. The L-proline-glycine ester saltof Cyclosporine A required purification. The salt was converted to thefree-base with sodium bicarbonate and purified by filtration throughsilica gel (eluting with acetone). The salt was then formed at lowtemperature with dilute anhydrous hydrochloric acid and dried.

Synthetic Sequence:

Synthesis of the N-(L-proline)-glycine and N-(L-Lysine)-glycine estersof Cyclosporine A a) pyridine; b) NaN₃, DMF; c) SnCl₂, methanol; d)boc-L-lysine, EDC; e) boc-L-proline, EDC; f) HCl, Et₂O ExperimentalSection

The synthesis of SPI0022 and SPI0023 was conducted in batches. Generallya small-scale experiment was performed first followed by a larger batch.Reagents mentioned in the experimental section were purchased at thehighest obtainable purity from Aldrich, Acros, or Bachem, except forsolvents, which were purchased from either Fisher Scientific orMallinkrodt. The Cyclosporine A (USP grade) used in these procedures wasprovided by Signature Pharmaceuticals, Inc.

Cyclosporine A (15.01 g, 0.0124 moles) was dissolved in anhydrouspyridine (35 mL) at room temperature, under an argon atmosphere. Thesolution was cooled to 5° C. in an ice/water batch and chloroaceticanhydride (9.10 g, 0.053 moles) was added. After stirring for 10minutes, the ice bath was removed and the solution was allowed to stirunder an argon atmosphere at room temperature for 17 hours. After 17hours, diethyl ether (200 mL) was added. The ether was washed with water(2×100 mL) and dried for 1 hour over sodium sulfate (10 g). Afterfiltration and concentration under reduced pressure, the remainingyellow foam was dried under high vacuum (1 hour at room temperature) andpurified by flash chromatography on silica gel (200 g), eluting withheptane/acetone (2:1). After combining and concentrating the productcontaining fractions, the remaining light yellow foam (14.8 g) waspurified a final time by crystallization from hot diethyl ether (140mL). After cooling (−10° C., 2 hours), filtration, and drying under highvacuum, the procedure generated the chloroacetate ester of CyclosporineA SPI001201 as a white solid (14.0 g, 88.3% yield).

Cyclosporine A chloroacetate ester

¹H NMR (300 MHz, CDCl₃):

δ=8.50 (d, 1H, J=9.6 Hz), 7.95 (d, 1H, J=6.6 Hz), 7.46 (d, 1H, J=9.0Hz), 7.40 (d, 1H, J=7.8 Hz), 5.35-4.52 (m, 15H), 4.37 (t, 1H, J=7.2 Hz),4.12 (d, 1H, J=14.7 Hz), 3.89 (d, 1H, J=14.7 Hz), 3.45-3.0 (m, 15H),2.8-2.5 (m, 6H), 2.5-1.5 (m, 16H), 1.5-0.7 (m, 53H).

¹³C NMR (75 MHz, CDCl₃):

δ=173.78, 173.37, 172.86, 172.61, 171.28, 171.18, 170.91, 170.79,168.78, 167.64, 167.18, 128.77, 126.68, 75.46, 65.95, 58.89, 57.47,55.80, 55.31, 54.86, 54.34, 50.19, 48.91, 48.35, 48.02, 44.80, 40.96,39.44, 37.07, 35.93, 33.85, 33.25, 32.40, 31.74, 31.50, 30.38, 30.12,29.82, 29.53, 25.13, 24.92, 24.78, 24.40, 23.99, 23.75, 22.85, 21.94,21.41, 21.25, 20.84, 19.85, 18.79, 18.32, 17.89, 17.82, 15.46, 15.24,10.08.

The chloroacetate ester of Cyclosporine A SPI001201 (10.10 g, 7.89mmole) was dissolved in anhydrous N,N-dimethlformamide (30 mL) at roomtemperature. Sodium azide (2.15 g, 33.0 mmole) was added. The mixturewas allowed to stir at room temperature for 24 hours in the dark, underan argon atmosphere. After 24 hours, diethyl ether (150 mL) was addedand the precipitate was filtered. The ether was washed with water (2×100mL), dried over sodium sulfate (15 g) for 30 minutes, filtered, andconcentrated under reduced pressure. The remaining white solid was driedunder high vacuum for 1 hour at room temperature. The experimentproduced the azidoacetate ester of Cyclosporine A SPI001202 (9.90 g, 97%yield) as a white solid, which was used without further purification.

Cyclosporine A azidoacetate ester

¹H NMR (300 MHz, CDCl₃):

δ=8.48 (d, 1H, J=9.3 Hz), 7.95 (d, 1H, J=6.9 Hz), 7.45 (d, 1H, J=9.0Hz), 7.39 (d, 1H, J=7.8 Hz), 5.5-4.5 (m, 15H), 4.31 (t, 1H, J=6.6 Hz),4.04 (d, 1H, J=17.3 Hz), 3.53 (d, 1H, J=17.3 Hz), 3.45-3.0 (m, 15H),2.8-2.5 (m, 6H), 2.5-1.5 (m, 16H), 1.5-0.7 (m, 53H).

¹³C NMR (75 MHz, CDCl₃):

δ=173.76, 173.32, 172.82, 172.53, 171.13, 170.89, 170.76, 170.69,169.70, 168.20, 167.49, 128.63, 126.61, 74.96, 58.91, 57.39, 55.56,55.21, 54.80, 54.23, 50.14, 48.99, 48.23, 48.24, 47.93, 44.71, 40.89,39.33, 39.22, 37.02, 35.83, 33.81, 32.96, 32.31, 31.67, 31.42, 30.31,30.09, 29.76, 29.47, 25.08, 24.92, 24.84, 24.67, 24.51, 24.40, 23.94,23.82, 23.71, 21.85, 21.33, 21.25, 20.82, 19.79, 18.71, 18.25, 17.92,17.81, 15.17, 10.03.

The azidoacetate ester of Cyclosporine A SPI001202 (9.80 g, 7.62 mmole)was dissolved in methanol (250 mL) at room temperature. Water (40 mL)was added followed by tin (II) chloride (5 g, 26.3 mmole). The solutionwas allowed to stir for 1 hour at room temperature when an additionalquantity of tin (II) chloride (4 g, 21.0 mmole) was added. The solutionwas allowed to stir for an additional 2 hours at room temperature. Water(200 mL) containing ammonium hydroxide (40 mL, 29%) was added. Afterfiltration, the solution was concentrated (to 200 mL) under reducedpressure. The remaining aqueous solution was extracted with ethylacetate (2×200 mL). The ethyl acetate fractions were combined, driedover sodium sulfate (20 g), filtered and concentrated under reducedpressure. The remaining clear foam was purified by filtration throughsilica gel (150 g), eluting with dichloromethane/methanol (20:1). Theprocedure generated the glycine ester of Cyclosporine A as a clear,solid foam (8.54 g, 89% yield).

Glycine ester of Cyclosporine A

¹H NMR (300 MHz, CDCl₃):

δ=8.60 (d, 1H, J=9.6 Hz), 8.06 (d, 1H, J=6.9 Hz), 7.53 (d, 1H, J=8.4Hz), 7.51 (d, 1H, J=6.6 Hz), 5.7-4.52 (m, 15H), 4.41 (t, 1H, J=6.9 Hz),3.5-3.0 (m, 17H), 2.82-2.5 (m, 8H), 2.5-1.5 (m, 16H), 1.5-0.7 (m, 53H).

¹³C NMR (75 MHz, CDCl₃):

δ=174.10, 173.67, 173.23, 172.72, 172.55, 171.18, 171.10, 170.73,170.61, 169.68, 167.77, 128.82, 126.42, 73.83, 58.57, 57.32, 55.99,55.20, 54.74, 54.31, 50.08, 48.82, 48.28, 47.90, 44.70, 43.81, 40.74,39.33, 39.24, 37.02, 35.84, 33.72, 33.07, 32.39, 31.72, 31.41, 30.25,29.98, 29.74, 29.51, 25.05, 24.81, 24.73, 24.54, 24.31, 23.91, 23.78,23.68, 21.86, 21.33, 21.25, 20.68, 19.76, 18.74, 18.24, 17.94, 17.79,15.18, 10.03.

The glycine ester of Cyclosporine A (SPI001203, 2.0 g, 1.59 mmole) wasdissolved in anhydrous dichloromethane (25 mL) with boc-L-lysine (1.31g, 3.78 mmole) and EDC (0.75 g, 3.9 mmole), under an argon atmosphere atroom temperature. The boc-L-lysine was prepared from thedicyclohexylamine salt (2.0 g in 50 mL ether) by extraction with coldpotassium hydrogen sulfate solution (1 g in 50 mL water) followed bycold water (2×50 mL). The ether containing the boc-L-lysine was driedover sodium sulfate (5 g), filtered, concentrated and dried under highvacuum for one hour at room temperature. A few crystals of DMAP wereadded to the mixture of EDC, boc-L-lysine, and the glycine ester ofCyclosporine A and the solution was allowed to stir for 4 hours at roomtemperature. The dichloromethane solution was extracted with DIUF water(50 mL), 5% sodium bicarbonate solution (50 mL), and with DIUF water (50mL). After drying over sodium sulfate (10 g), the dichloromethanesolution was filtered and concentrated under reduced pressure. Theremaining white foam (3.01 g) was purified by flash columnchromatography on silica gel (50 g), eluting with heptane/acetone (2:1).The product containing fractions were combined; concentrated underreduced pressure, and dried under high vacuum. The purified protectedintermediate (2.34 g white solid, 92.8% yield) was placed in a flaskunder an argon atmosphere, which was cooled in an ice-water bath. Coldanhydrous 2 M hydrochloric acid in diethyl ether (20 mL) was added andthe solution stirred for 8 hours (at 5° C.). The mixture was slowlyallowed to warm to room temperature overnight. After stirring for atotal of 20 hours, the flask was cooled again in an ice-water bath for30 minutes. The product was filtered and dried under high vacuum for 1hour at room temperature and then at 50° C. for 4 hours. The experimentproduced Cyclosporine A N-(L-lysine)-glycine ester, dihydrochloridetrihydrate (SPI0022, 1.59 g, 73.9% yield) as a white solid.

¹H NMR (300 MHz, CDCl₃, NMR data is for the free base):

δ=8.58 (d, 1H, J=9.3 Hz), 8.04 (d, 1H, J=6 Hz), 7.80 (d, 1H, J=6 Hz),7.49 (d, 2H, J=8.4 Hz), 5.70-4.6 (m, 17H), 4.41 (m, 1H), 4.28 (dd, 1H,J=17, 7.2 Hz), 3.67 (d, 1H, J=17Hz), 3.46 (s 3H), 3.4-2.8 (m, 16H),2.8-2.5 (m, 8H), 2.5-1.35 (m, 24H), 1.5-0.7 (m, 50H).

¹³C NMR (75 MHz, CDCl₃, NMR data is for the free base):

δ=175.23, 173.77, 173.34, 172.75, 172.63, 171.34, 171.22, 170.94,170.84, 170.91, 169.89, 169.70, 128.74, 126.67, 74.41, 58.82, 57.43,55.91, 55.21, 54.81, 54.42, 50.17, 48.89, 48.31, 47.98, 44.78, 41.92,40.82, 40.69, 39.44, 39.32, 27.19, 35.91, 34.88, 33.71, 33.25, 33.12,32.44, 31.83, 31.50, 30.38, 30.06, 29.81, 29.55, 25.14, 24.90, 24.52,24.43, 24.00, 23.76, 21.93, 21.42, 21.29, 20.81, 19.84, 18.82, 18.32,17.96, 17.86, 15.21, 10.10.

CHN Analysis:

Calculated for C₇₀H₁₂₈Cl₂N₁₄O₁₅-3H₂O: C, 55.50, H, 8.92, and N, 12.74.found: C, 58.28, H, 8.98, and N, 13.16.

HPLC Analysis:

99.60% purity; r.t.=14.763 min.; 80% acetonitrile/20% Tris base in DIUFwater; 1 mL/min; 60C; Synergi Hydro RP, 4 u column (serial #163383-7),4.6×250 mm; 20 ul; UV=210 nm.

Melting point: 196.0-198° C. (uncorrected)

The glycine ester of Cyclosporine A (SPI001203, 7.50 g, 5.95 mmole) wasdissolved in anhydrous dichloromethane (50 mL) with boc-L-proline (2.56g, 11.90 mmole) and EDC (2.28 g, 11.9 mmole), under an argon atmosphereat room temperature. A few crystals of DMAP were added to the mixture ofEDC, boc-L-proline, and the glycine ester of Cyclosporine A and thesolution was allowed to stir for 3 hours at room temperature. Thedichloromethane solution was extracted with DIUF water (50 mL), 5%sodium bicarbonate solution (2×50 mL), and with DIUF water (50 mL).After drying over sodium sulfate (10 g), the dichloromethane wasfiltered and concentrated under reduced pressure. The remaining whitefoam (9.50 g) was purified by flash column chromatography on silica gel(150 g), eluting with heptane/acetone (2:1 followed by 1:1). The productcontaining fractions were combined, concentrated under reduced pressure,and dried under high vacuum (7.94 g white solid, 91.7% yield) for 10minutes at room temperature.

The purified protected intermediate (6.46 g) was placed in a flask underan argon atmosphere, which was cooled in an ice-water bath. Coldanhydrous 2 M hydrochloric acid in diethyl ether (150 mL) was added andthe solution stirred for 8 hours (at 5° C.). The mixture was slowlyallowed to warm to room temperature overnight. After stirring for atotal of 20 hours, the flask was cooled again in an ice-water bath for30 minutes. The product was filtered and dried under high vacuum for 30minutes at room temperature. The Cyclosporine A N-(L-proline)-glycineester, hydrochloride (5.17 g, 84.6% yield, and 90% purity by HPLC) wasconverted to the free base by dissolving the salt in DIUF water (25 mL)that contained sodium bicarbonate (1 g). The free base was extractedwith dichloromethane (3×25 mL), which was dried over sodium sulfate (5g), filtered and concentrated. The remaining off-white solid (5 g) waspurified by filtration through silica gel (100 g), eluting with acetone.The product containing fractions were combined, concentrated underreduced pressure, and dried under high vacuum for 30 minutes at roomtemperature. The hydrochloride salt was regenerated by dissolving thefree base (3.8 g) in diethyl ether (25 mL) and adding it to anhydrous 2Mhydrochloric acid (5 mL) in heptane (50 mL), while cooling in anice-water bath. After 20 minutes at 5° C., the white solid was filteredand dried under high vacuum for hours at room temperature. Theexperiment produced Cyclosporine A N-(L-proline)-glycine ester,hydrochloride (SPI0023, 3.8 g) as a white solid.

¹H NMR (300 MHz, CDCl₃):

δ=14.20 (br s, 2H), 8.62 (d, 1H, J=10 Hz), 8.06 (d, 1H, J=6.9 Hz), 7.61(d, 1H, J=8.1 Hz), 7.48 (d, 1H, J=9Hz), 5.70-5.50 (m, 3H), 5.40-4.60 (m,12H), 4.37 (m, 1H), 4.20 (d, 1H, J=18 Hz), 3.97 (d, 1H, J=18 Hz), 3.70(m, 1H), 3.45 (s, 3H), 3.23-3.08 (m, 12H), 2.66 (s, 3H), 2.60 (s, 3H),2.50-1.80 (m, 15H), 1.78-1.20 (m, 15H), 1.15-0.66 (m, 46H).

¹³C NMR (75 MHz, CDCl₃):

δ=174.15, 173.49, 172.67, 172.59, 171.86, 171.20, 171.13, 171.02,170.83, 169.68, 168.77, 167.55, 128.30, 127.10, 80.09, 75.58, 62.65,59.35, 57.36, 55.53, 55.30, 54.78, 54.35, 53.60, 50.25, 50.09, 48.92,48.18, 48.12, 44.62, 40.59, 40.02, 39.43, 39.30, 37.13, 35.88, 33.74,33.07, 32.19, 32.01, 31.86, 31.50, 31.43, 30.43, 29.93, 29.72, 29.30,29.16, 27.56, 26.04, 25.00, 24.86, 24.74, 24.39, 20.96, 19.81, 18.71,18.26, 18.09, 17.85, 17.79, 15.09, 14.30, 10.00.

CHN Analysis:

Calculated for C₆₉H₁₂₂ClN₁₃O₁₄: C, 59.48, H, 8.83, and N, 13.07. found:C, 59.84, H, 9.02, and N, 12.65.

HPLC analysis:

99.59% purity; r.t.=10.613 min.; 85% acetonitrile/15% Tris base in DIUFwater; 1.2 mL/min; 60C; Synergi Hydro RP, 4 u column (serial #163383-7),4.6×250 mm; 20 ul;

UV=210 nm.

Melting point: 197.0-199° C. (uncorrected)

The amino acid derivatives of cyclosporin of the present invention areeffective in treating diseases or conditions in which macrocyclicimmunosuppressants normally are used. These derivatives are transformedwithin the body to release the active compound and enhances thetherapeutic benefits of the macrocyclic immunosuppressants by reducingor eliminating biopharmaceutical and pharmacokenetic barriers associatedwith each of them. However it should be noted that these derivativesthemselves will have sufficient activity without releasing any activedrug in the mammals. Since the derivatives are more soluble in waterthen cyclosporine or other macrocyclic immunosuppressants, it does notneed to be associated with a carrier vehicle, such as alcohol or castoroil which may be toxic or produce unwanted side reactions. Moreover,oral formulations containing the derivatives of the derivatives areabsorbed into the blood and are quite effective.

Thus, the derivative of cyclosporin of the present invention enhancesthe therapeutic benefits by removing biopharmaceutical andpharmacokenetic barriers of existing drugs.

Furthermore, the derivatives are easily synthesized in high yields usingreagents which are readily and commercially available.

Animal Effiacy Study Results for Cyclosporine Derivatives:

A simple method to test the efficacy of cyclosporine A and its analogs,Cyclosporine-glycine-proline (Cyclosporine GP) ester,Cyclosporine-glycine-lysine (Cyclosporine GL) ester andCyclosporine-glycine (Cyclosporine G) ester is to treat ulcerativecolitis induced by administering 5% Dextran Sodium Sulfate (DSS) adlibitum to laboratory mice. This model has been used consistently withmost immunosuppressants to demonstrate both actual treatment afterinduction of colitis, and also prophylactic treatments. Results fromboth types of studies are shown below.

Calculation of Disease Activity Index (DAI)

Percent animal body weight loss, stool consistency and occult blood atthe end of the study will be taken into consideration to calculateDisease Activity Index (“DAI”) of each animal. The following scoringsystem outlined in Table 30 was for the percent body weight loss, stoolconsistency and occult blood:

TABLE 30 Score Weight loss (%) Stool consistency Blood in feces 0 0 orgain Normal Negative 1   1-4.9 Soft +/− 2 5.0-9.9 Mixed (soft & +Diarrhea) 3 10-15 Diarrhea ++ 4 >15 Bloody Diarrhea +++ (Gross blood)The scores for each parameter for each mouse at the end of study areadded together and divided by three (number of observations) todetermine DAI. The mean group DAI is the determined. A score of 3 wasassigned for any animal that died during the testing.Results from Prophylactic Treatment:

In this study, DSS administration is coupled with administration ofeither test or reference drug from day 1. No of mice used were 10 ineach group:

TABLE 31 Drug Mean DAI Vehicle 1.500 Cyclosporine A (Ref) 1.400Cyclosporine-G 1.531 Cyclosporine-GL 1.100 Cyclosporine-GP 1.062

While Cyclosporine G didn't perform as expected in this model,Cyclosporine GP had statistically significant performance compared toCyclosporine A.

Results from Disease Treatment Study:

Much more dramatic results were obtained when the mice were initiallytreated with DSS to induce colitis. Once clear symptoms of colitis wereseen, DSS was replaced by drinking water, and treatment started withtest and reference drugs, including vehicle control. All results aremean of 10 mice. The mean DAI os tabulated hereinbelow in Table 32:

TABLE 32 Drug Mean DAI Vehicle 1.832 Cyclosporine A (Ref) 0.300Cyclosporine-G 0.100 Cyclosporine-GL 0.532 Cyclosporine-GP 0.066

Clearly Cyclosporine G and Cyclosporine-GP showed statisticallysignificant results, significant improvement over Cyclosporine referencedrug. Notice in the treatment study, 5 out of 10 mice died in thevehicle control, 1 died with cyclosporine-GL treatment, and none died inthe cyclosporine-A, Cyclosporine-G and Cyclosporine-GP treated groups.

Development of Cyclosporine GP Ophthalmic Preparation:

Clear, aquous solutions of the Cyclosporine GP was prepared bydissolving sufficient quantities of Cyclosporine GP in distilled water,and pH adjusted to around 5 to produce 0.2% w/v concentrations.Allowable quantities of HCl, and alcohol as per US FDA ophthalmicadditive content regulations were added to keep the mixture stable andsolubilized in the ophthalmic preparation. The resulting clear solutionwas stable for 3 months at room temperature. This solution appearedpharmaceutically elegant than currently available formulations such asRestasis® and other cloudy preparations using cremaphor or castor oil tomake emulsion of cyclosporine in water.

As indicated hereinabove the solubility of the amino acid derivatives ofthe macrocyclic immunosuppressent with an OH group functionality groupthereon with which the amino acid can form an ester linkage issignificantly enhanced in aqueous solution relative to the correspondingmacroscopic immunosuppressants which are not linked to the amino acids.For example, the solubility of cyclosporine in water is approximately 30microgram/ml (US patent publication 20040138108). The solubility ofCyclosporine-glycine-proline ester in aquous solution at roomtemperature was 60 times that of Cyclosporine A in water, and equaledapproximately 2000 micrograms/ml.

VI. Valproic Acid Esters

Valproic acid (2-Propylpentanoic acid) is low molecular weightcarboxylic acid derivative which is widely used as an anti-convulsiveagent, useful in the treatment of epilepsy and also possessvasodilatation activity in the brain to relieve migraine headaches. Itis administered orally to control epileptic episodes in humans and alsoalleviate severe pain associated with migraine headaches.

Valproic acid has been shown to have a large number of therapeuticapplications, which are quite varying and somewhat surprising. Forexample, in addition to its efficacy in the treatment of epilepsy andmigraine headaches, it has been shown to be effective in the treatmentof certain psychiatric illnesses, such as bipolar disorder, moodstabilization, control of aggression, impulsivity in personalitydisorder, agitation in dementia, and has also been of use as adjuncttherapy in the treatment of post traumatic stress disorder (PTSD).

Mechanism of Action:

In spite of being used in the treatment of epilepsy for a number ofyears, the exact mechanism of action of Valproic acid is still unknown.It has been postulated that it exerts its action by increasingconcentration of gamma-amino butyric acid (GABA) in the brain.Gamma-amino butyric acid is a neurotransmitter, a chemical that nervesuse to communicate with one another.

Valproate is the drug of choice in myoclonic epilepsy, with or withoutgeneralized tonic-clonic seizures, including juvenile myoclonic epilepsyof Janz that begins in adolescence or early adulthood. Photosensitivemyoclonus is usually easily controlled. Valproate also is effective inthe treatment of benign myoclonic epilepsy, postanoxic myoclonus, and,with clonazepam, in severe progressive myoclonic epilepsy that ischaracterized by tonic-clonic seizures as well. It also may be preferredin certain stimulus-sensitive (reflex, startle) epilepsies.

Although Valproate may be effective for infantile spasms, it isrelatively contraindicated in children whose spasms are due tohyperglycinemia or other underlying metabolic (mitochondrial)abnormalities. In general, atonic and akinetic seizures in patients withLennox-Gastaut syndrome are difficult to control, but Valproate is thedrug of choice for treatment of mixed seizure types. Since this drug hasbeen useful in some patients who are refractory to all otherantiepileptic drugs, it may warrant a trial in nearly all nonresponsivepatients regardless of seizure type.

In spite of it usefulness, hepatotoxicity may be fatal, but isidiosyncratic and not preventable by routinely monitoring liver enzymes.Hepatotoxicity occurs in very young children, most often those onmultiple anticonvulsants. Valproate-induced cytopenias may bedose-related and warrant monitoring of complete blood counts duringtherapy. Encephalopathy with hyperammonemia without liver function testabnormalities may occur. Pregnant women in their first month are at riskfor neural tube defects.

Valproic acid is a low molecular weight liquid with characteristic odor.Taken orally, it has unpleasant taste and can severely irritate mouthand throat. In order to convert Valproic acid into a solid dosage formconvenient for oral administration, a number of derivatives withcovalent and ionic bond with the carboxylic acid have been made. Asimple sodium salt of Valproic acid, resulting in Valproate sodium isavailable as a solid. However a stable coordination complex, know asDivalproex sodium was formed by partial neutralization of two moleculesof Valproic acid with one atom of sodium. This product is the mostwidely available commercial Valproic acid hemisalt. It is marketed byAbbott Laboratories in the USA under the brand name Depakote®. Depakote®is also available in extended release formulation for oraladministration.

A significant disadvantage of Valproic acid is that in liquid form, itis difficult to administer. Furthermore, administration of Valproic acidin different forms does not uniformly produce desired bioavailability.For example, the overall bioavailability of Valproate from Valproicacid, its sodium salt, Divalproex®, and their extended releaseformulations are not quite interchangeable. Since continuous monitoringof the plasma profile of a patient to whom Valproic acid is administeredis essential, any change in plasma concentration due to changes in theformulation can adversely affect the overall therapeutic outcome.

The amino acid derivatives of Valproic acid improve the therapeuticeffectiveness, uniform blood profile, develop pharmaceutically elegantformulation and reduce first pass metabolism, exhibited by valproic acidalone.

Until now there has been no pharmaceutical preparation available in themarket that can deliver Valproic acid without harmful side effects. Thepresent invention, however, has produced a number of water soluble,non-toxic derivatives of Valproic acid which are suitable for deliveringValproic acid consistently in the body without any harmful side effectsand without the needs for expensive additives and excipients.

Accordingly, in one aspect, the present invention is directed to a classof amino acid derivatives of Valproic acid or acylating derivativethereof. The derivative consists of the hydroxyl group of an amino acidesterified to the free carboxyl group present on the Valproic acidmolecules. In another embodiment, the amine group of the amino acid isreacted with COOH of the valproic acid or acylating derivative thereofto form an amide linkage.

More specifically, an embodiment of the present invention is directedto, the compounds of the formula

or pharmaceutically acceptable salts thereof wherein R is either NH-AAor O-AA and AA is an amino acid less an amino group in NHAA or less anhydroxy group in O-AA, in which either an amine group or the hydroxylgroup is reacted with the carboxylic acid group of Valproic Acid.

Recently A small proof of concept study (Depletion of latent HIV-1infection in vivo: a proof-of-concept study Lehrman G, Hogue I B, PalmerS, Jennings C, Spina C A, Wiegand A, Landay A L, Coombs R W, Richman DD, Mellors J W, Coffin J M, Bosch R J, Margolis D M. The Lancet—Vol.366, Issue 9485, 13 Aug. 2005, Pages 549-555) demonstrated that ValproicAcid might reduce the pool of dormant HIV infected cells in the body.

As part of its life cycle, HIV needs to insert its genetic material intothe DNA of the human CD4 T-cells which it infects. These incorporatedgenes of the virus are then used as template for the production of newvirus particles in active cells. This process leads to the cells beingover-activated, eventually causing most of them to commit suicide or‘apoptosis’ and the CD4 cell counts to fall.

However, a small proportion of the HIV-infected cells do not die, butbecome dormant within the lymph node or other areas of the body. Sincethey are not actively producing new HIV particles, the genes of thevirus remain hidden within the nuclei of these cells. These cells, whichare distributed throughout the body, are highly stable and long-lasting,but remain ready to start pumping out new HIV particles if they becomeactivated by stimulation of the immune system.

Currently available anti-retroviral drug combinations (known as AIDScocktail) can reduce HIV to undetectable levels in the blood, but theycan only do so by preventing HIV from making copies of itself, or‘replicating’ in the body. Anti-retroviral drugs typically can notremove HIV's genes that are integrated into the human cells' DNA,leaving the reservoir of the HIV in latent infected cells untouched.

Valproic Acid (VPA) which is currently used worldwide for the treatmentof bipolar disorders and epilepsy is an inhibitor of a cellular enzymecalled histone deacetylase 1 (HDAC1). This enzyme has been shown to becrucial in keeping HIV's gene hidden within the host cells' DNA.

In the test tube, studies have shown that VPA can stimulate the releaseof the HIV from resting infected T-Cells. Consequently, scientists aretrying to find out whether giving the drug to HIV-positive patientscould stimulate the release of resting cells to start producing andexpressing HIV particles and thus eliminate the hidden stores of HIVfrom the body. Once the viral particles are exposed to anti-retroviraldrugs, the disease is cured since there are no hidden reservoirs in thebody in the short run.

Problems with VPA Therapy:1. A small proof of concept study explained above was done in a limitednumber of patients (4 patients), and it was not a proof of efficacystudy. When these 4 patients were treated for 18 weeks with Valproicacid (“VPA”), the investigators saw a decline in resting cellscontaining HIV genes between 68 to 84%, and in the 4^(th) patient, hadsmaller reduction to 29%.2. VPA is not without its severe side effects and adverse reactions. FDAhas mandated boxed bold letter warnings for this drug to be included inall product inserts. Serious box warning include hepatotoxicity,teratogenicity, pancreatitis.3. Other serious side effects include urea cycle disorders (UCD),somnolence in the elderly, thrombocytopenia, hyperammonemia, birthdefects, mania, and various GI, Nervous system, respiratory system, skindisorders and weight gain.4. While VPA has shown to be carcinogenic and mutagenic in animalmodels, such effects in humans are unknown.5. Dr. Robert Siliciano from the Johns Hopkins University is of theopinion that “its extremely unlikely that this approach would work”, whois one of the scientists who discovered dormant infection of the HIV inthe mid-1990s.6. According to Dr. Siliciano, 99.9999% of the hidden HIV infection hasto be “kicked” out of the dormant human CD4 T-cells. In the proof ofconcept study, it was shown that reduction levels varied between 29 to84%, far short of the 99.9999% demanded by Dr. Siliciano.

Solution to the Problem

The amino acid derivatives of valproic acid represent the solution tothe problem. They are effective in expelling the virus, e.g., HIV virusfrom the human immune cell reservoirs.

Table 33 lists as examples, 6 amino acid derivatives of valproic acideffective in expelling the virus:

TABLE 33 No Drug Mol. Wt 1 Valproic Acid L-Threonine Ester 281.78 2Valproic Acid Serine Ester 267.76 3 Valproic Acid Serine Amide Nitrate276.29 4 Valproic Acid Tyrosine Ester 343.85 5 Valproic AcidHydroxyproline Amide 257.33 6 Valproic Acid Hydroxyproline Ester 257.33

The present invention is also directed to a pharmaceutical compositioncomprising a therapeutically effective amount of the an amino acidderivative of Valproic acid and a pharmaceutical carrier therefor.

In another embodiment, the present invention is directed to a method oftreating a patient in need of Valproic acid therapy, which methodcomprises administering to said patient an effective amount of the aminoacid derivative of the Valproic acid.

In a further embodiment, the present invention is directed to a methodof converting liquid Valproic acid into a solid powder by reacting thecarboxyl functionality of the Valproic acid with either the amine orhydroxyl functionality of an amino acid or acylating derivative thereofand isolating the product(s) thereof.

In a still further embodiment, the present invention is directed to amethod of substantially and in a therapeutically efficacious manner,reducing or eliminating the potential first pass metabolism therebyimproving the consistent therapeutic effect by administering to apatient an amino acid derivative which comprises reacting the COOHfunctionality of the Valproic acid molecule with either the NH₂ or OHfunctionality of the amino acids to form an ester or amide covalentbond, respectively, and isolating the product thereof and administeringsaid product to the patient.

The present inventor has found that when naturally occurring amino acidsare esterified to Valproic acid, the resulting derivatives arepharmaceutically elegant free flowing powders, and are rapidly absorbedinto the body and release non-toxic amino acids upon cleavage in thebody and require none of the emulsifiers, additives and other excipientsassociated with volproic acid.

Furthermore, the amino acid derivatives of Valiproic acid are effectiveanti-epileptics and exhibit such effect intact. Thus the current aminoacid derivatives are effective anti-epileptics and useful in thetreatment of a number of psychiatric illnesses and exhibit suchpotential with or without releasing the active parent drug.

The amino acid derivative of Valproic acid exhibits a bulk density whichis much higher than the corresponding sodium salts, and they aresuitable for compacting large weight tablets and capsules. Furthermore,the amino acid derivatives of Valproic acid do not exhibit bitter tasteand unusual odor of the Valproic acid.

As indicated hereinabove, the amino acid derivatives are effectiveanti-epileptics with or without releasing Valproic acid. However, whenadministered in vivo, the amino acid derivative release in vivo theactive drug with all its pharmacological and psychoactive properties.

In conclusion, the amino acid derivative of Valproic acid clearlyprovides a number of advantages over Valproic acid. For example, theyare less toxic than the corresponding valproic acid when administered invivo, the valproic acid amino acid derivatives may be metabolized tocleave the amino acid moiety from the valproic acid, which arenon-toxic. This results in a high therapeutic index. Secondly, the aminoacid derivatives may or may not cleaved in the body to release Valproicacid. The amino acid derivative of valproic acid exhibits the sameutility as valproic acid itself. Furthermore, due to their high watersolubility, they can be easily administered by either forming an in-situsolution just before IV administration using lyophilized sterile powderor providing the drug in solution in prefilled syringe or bottles forinfusion. The amino acid esters are more stable than Valproic acid sincethe COOH group in Valproic acid is blocked so as to minimize anyreaction with bases. Thus the amino acid derivatives of Valproic acidare more effective than Valproic acid itself without the toxicity andother pharmaceutical problems associated with current marketedformulations of valproic acid.

The procedures for the synthesis of the L-serine, L-threonine, andL-hydroxyproline esters of valproic acid (2-propylpentanoic acid) areoutlined in Synthetic Sequence section and is exemplary for thepreparation of the various derivatives of the present invention. Thecomplete procedure and analytical data is given in the ExperimentalSection. In general, valproic acid (2-8 g, in batches) was coupled withthe N-benzyloxy/benzyl ester protected amino acids using EDC in thepresence of a catalytic amount of DMAP. Once the reactions were complete(20 hours at room temperature), the mixture was extracted with DIUFwater, dried over sodium sulfate, and concentrated under reducedpressure. The crude material was either used directly for thedeprotection step or purified by column chromatography. The proceduregenerated the protected amino acid esters of valproic acid in yieldsranging from 72% to 92%. The protecting groups were removed byhydrogenation (30 psi H₂) in the presence of 10% palladium on carbon.The amino acid esters of valproic acid were extracted away from thepalladium catalyst with ethanol, concentrated, and dried. The finalsalts were formed by acidification with hydrochloric acid. The crudesalts (yields ranging from 57% to 92%) were then purified by the methodsdescribed in the Experimental Section.

Synthetic Sequence:

Synthesis of the L-serine, L-threonine, and L-hydroxyproline esters ofValproic acid a) EDC, DMAP, CH₂Cl₂; b) H₂, 10% Pd/C, EtOH, EtOAc; c) HClExperimental Section

The synthesis of SPIC001, SPIC002 and SPIC003 were conducted in one ortwo batches. Reagents mentioned in the experimental section werepurchased at the highest obtainable purity from Lancaster,Sigma-Aldrich, Acros, or Bachem, except for solvents, which werepurchased from either Fisher Scientific or Mallinkrodt.

1) SPIC001 2-Propylpentanoic acid 2(S)-amino-2-carboxy-ethyl ester,hydrochloride

-   -   (L-Serine-valproic acid ester, hydrochloride)

A mixture of 2-propylpentanoic acid (valproic acid, 6.48 g, 44.93mmole), N-carbobenzyloxy-L-serine benzyl ester (Z-Ser-OBzl, 14.80 g,44.93 mmole), EDC (8.61 g, 44.91 mmole), and DMAP (549 mg, 4.49 mmole)in anhydrous dichloromethane (50 mL) was stirred under an argonatmosphere at room temperature for 20 hours. After 20 hours, thedichloromethane was washed with water (3×50 mL), dried over magnesiumsulfate (5 g), filtered and concentrated under reduced pressure. Theremaining colorless oil (20.87 g) was purified by column chromatographyon silica gel (150 g, 0.035-0.070 mm, 6 nm pore diameter), eluting withhexanes/ethyl acetate (3:1). After concentration of the productcontaining fractions under reduced pressure and drying under high vacuumuntil the weight was constant, the experiment produced the protectedL-serine-valproate ester SPIC00101 (18.9 g, 92% yield) as a colorlessoil.

¹H NMR (300 MHz, DMSO): δ=7.96 (1H, d, J=8.1 Hz), 7.35 (10H, m), 5.14(2H, s), 5.05 (2H, s), 4.51 (1H, m), 4.29 (2H, m), 2.29 (1H, m),1.50-1.25 (4H, m), 1.25-1.10 (4H, m), 0.80 (6H, t, J=6.6 Hz).

¹³C NMR (75 MHz, DMSO): δ=174.88, 169.15, 155.85, 136.58, 135.45,128.26, 128.18, 127.47, 127.71, 127.57, 66.32, 65.66, 62.47, 53.09,44.20, 33.86, 33.79, 19.95, 13.85.

The protected L-serine-valproate ester SPIC00101 (18.9 g, 41.48 mmole)was dissolved in ethanol (60 mL) and ethyl acetate (60 mL) at roomtemperature and added to a Parr bottle (500 mL) that contained 10%palladium on carbon (3.0 g, 50% wet) under a nitrogen atmosphere. Thenitrogen atmosphere was replaced with hydrogen gas (30 psi). After 4hours of shaking, additional palladium catalyst (1.0 g) in ethanol\ethylacetate (1:1, 100 mL) was added and the reaction mixture shook overnightunder hydrogen gas (30 psi) at room temperature. After 24 hours thecatalyst was removed by filtration through a thin layer of activatedcarbon. The ethanol and ethyl acetate were concentrated under reducedpressure at room temperature. After drying under high vacuum, theremaining solids were acidified with hydrochloric acid in diethyl ether(2M, 24.6 mL). The mixture was stored in a refrigerator for two hoursbefore filtration and washing with additional cold diethyl ether (10mL). After filtration, the remaining white solid was dried at roomtemperature under high vacuum until the product weight was constant (24hours). The experiment produced L-serine-valproic acid ester,hydrochloride SPIC001 (6.34 g, 57% yield) as a white solid.

¹H NMR (300 MHz, DMSO): δ=8.73 (br s, 3H), 4.47 (dd, 1H, J=12.9, 4.5Hz), 4.31 (dd, 2H, J=12.9, 3.6 Hz), 2.36 (m, 1H), 1.50 (m, 2H), 1.39 (m,2H), 1.20 (m, 4H), 0.84 (t, 6H, J=7 Hz).

¹³C NMR (75 MHz, DMSO): δ=174.67, 168.19, 61.84, 51.16, 44.12, 33.76,33.58, 20.07, 19.92, 13.97, 13.89.

HPLC Analysis:

98.49% purity; rt=4.767 min; Luna C18 5 u column (sn 167917-13); 4.6×250mm; 254 nm; 33% ACN/66% DIUF water; 35 C; 20 ul inj.; 1 ml/min; 20 mg/mLsample size; sample dissolved in mobile phase.

CHN Analysis:

calc.: C, 49.34; H, 8.28; N, 5.23. found: C, 49.22; H, 8.35; N, 5.24.

Melting point: 159-160° C.

2) SPIC002 4(R)-(2-Propyl-pentanoyloxy)-pyrrolidine-2(S)-carboxylic acidL-Hydroxyproline-valproic acid ester

A mixture of 2-propylpentanoic acid (valproic acid, 4.32 g, 30 mmole),N-carbobenzyloxy-L-hydroxyproline benzyl ester (Z-Hyp-OBzl, 10.66 g, 30mmole), EDC (5.74 g, 30 mmole), and DMAP (366 mg, 3 mmole) in anhydrousdichloromethane (30 mL) was stirred under an argon atmosphere at roomtemperature for 20 hours. After 20 hours, the dichloromethane was washedwith water (3×30 mL), dried over magnesium sulfate (5 g), filtered andconcentrated under reduced pressure. The remaining colorless oilSPIC00201 (11.95 g, 24.7 mmole, 82.4% yield) was used withoutpurification.

¹H NMR (300 MHz, CDCl₃): δ=7.29 (10H, m), 5.28-5.00 (5H, m), 4.55 (½H,t, J=8 Hz), 4.46 (½H, t, J=8 Hz), 3.80-3.60 (2H, m), 2.43-2.16 (3H, m),1.60-1.45 (2H, m), 1.40-1.32 (2H, m), 1.28-1.20 (4H, m), 0.86 (6H, m).

¹³C NMR (75 MHz, DMSO): δ=174.74, 171.40, 171.05, 153.79, 153.31,136.34, 136.20, 135.57, 135.38, 128.24, 128.13, 127.95, 127.87, 127.67,127.52, 127.28, 127.10, 72.29, 71.53, 66.34, 66.10, 57.66, 57.19, 52.27,51.89, 44.13, 40.33, 35.78, 34.79, 34.04, 33.92, 33.35, 20.00, 19.91,13.79, 13.73.

The protected L-hydroxyproline-valproate ester SPIC00201 (17.24 g, 35.79mmole) was dissolved in ethanol (50 mL) and ethyl acetate (100 mL) atroom temperature and added to a Parr bottle (500 mL) that contained 10%palladium on carbon (3.5 g, 50% wet) under a nitrogen atmosphere. Thenitrogen atmosphere was replaced with hydrogen gas (30 psi). After 15hours of shaking, the catalyst was removed by filtration through a thinlayer of celite and activated carbon. The ethanol and ethyl acetatemixture was concentrated under reduced pressure at room temperature.After drying overnight under high vacuum at room temperature, theexperiment produced L-hydroxyproline-valproic acid ester SPIC002 (9.2 g,99.8% yield) as a white solid. In order to remove trace impurities, thezwitterion was purified by reverse-phase column chromatography (50 g ODSsilica gel) in two batches. The zwitterion was placed on the column inDIUF water and eluted with mixture of DIUF water/methanol (2:1, 1:1,1:2, 100% methanol).

The product containing fractions were combined, concentrated underreduced pressure at 20° C. (or less), and dried under high vacuum atroom temperature until the weight was constant (24 hours, 6.4 g whitesolid recovered).

¹H NMR (300 MHz, CDCl₃): δ=12.40 (br s, 1H), 8.32 (br s, 1H), 5.28 (m,1H), 4.11 (t, 1H, J=7.2 Hz), 3.59 (m, 1H), 3.34 (br d, 1H, J=10.5Hz),2.50-2.22 (m, 3H), 1.62-1.50 (m, 2H), 1.50-1.32 (m, 2H), 1.32-1.19 (m,4H), 0.88 (t, 6H, J=7.2 Hz).

¹³C NMR (75 MHz, CDCl₃): δ=175.99, 173.35, 71.83, 59.56, 49.77, 45.08,36.19, 34.51, 20.87, 14.31.

HPLC Analysis:

99.20% purity; r.t.=7.228 min.; 70% DIUF water/30% acetonitrile; 1mL/min; 36.8C; Luna C18, 5 u column (serial #167917-13), 4:6×250 mm; 22ul injection; sample dissolved in mobile phase.

CHN Analysis:

calc.: C, 60.68; H, 9.01; N, 5.44. found: C, 60.58; H, 9.12; N, 5.48.

Melting point: 179.0-180.0° C.

3) SPIC003 2-Propyl-pentanoic acid2(S)-amino-2-carboxy-l(R)-methyl-ethyl ester, hydrochloride

-   -   (L-Threonine-valproic acid ester, hydrochloride)

A mixture of 2-propylpentanoic acid (valproic acid, 4.32 g, 30 mmole),N-carbobenzyloxy-L-threonine benzyl ester (Z-Thr-OBzl, 10.30 g, 30mmole), EDC (5.74 g, 30 mmole), and DMAP (366 mg, 3.0 mmole) inanhydrous dichloromethane (30 mL) was stirred under an argon atmosphereat room temperature for 20 hours. After 20 hours, the dichloromethanewas washed with water (3×30 mL), dried over magnesium sulfate (5 g),filtered and concentrated under reduced pressure. The remainingcolorless oil (13.44 g) was purified by column chromatography on silicagel (100 g, 0.035-0.070 mm, 6 nm pore diameter), eluting withhexanes/ethyl acetate (4:1).

After concentration of the product containing fractions under reducedpressure and drying under high vacuum until the weight was constant, theexperiment produced the protected L-threonine-valproate ester SPIC00301(12.65 g, 89.8% yield) as a colorless oil.

¹H NMR (300 MHz, CDCl₃): δ=7.40-7.05 (11H, m), 5.45 (1H, m), 5.17-5.02(4H, m), 4.53 (1H, d, J=9.6 Hz), 2.24 (1H, m), 1.58-1.40 (2H, m),1.40-1.15 (9H, m), 0.86 (6H, m).

¹³C NMR (75 MHz, DMSO): δ=174.24, 169.29, 156.48, 136.61, 135.34,128.26, 128.20, 127.74, 127.67, 127.58, 69.04, 66.33, 65.78, 57.62,44.50, 33.89, 33.80, 20.03, 19.91, 16.40, 13.87.

The protected L-threonine-valproate ester SPIC00301 (12.65 g, 26.9mmole) was dissolved in ethanol (50 mL) and ethyl acetate (50 mL) atroom temperature and added to a Parr bottle (500 mL) that contained 10%palladium on carbon (2.53 g, 50% wet) under a nitrogen atmosphere. Thenitrogen atmosphere was replaced with hydrogen gas (30 psi). After 20hours the catalyst was removed by filtration through a thin layer ofactivated carbon, washing with ethanol (25 mL). The ethanol and ethylacetate were concentrated under reduced pressure at room temperature.After drying under high vacuum, the remaining solids (6.13 g) wereacidified with hydrochloric acid (3.1 mL conc.) in DIUF water (50 mL).The solution was filtered a second time through activated carbon anddried overnight in a freeze-dryer. The experiment producedL-threonine-valproic acid ester, hydrochloride SPIC003 (6.52 g, 86.0%yield) as a white solid.

The combined batches of the L-threonine-valproic acid ester,hydrochloride SPIC003 (8.8 g) were purified by crystallization formacetonitrile. After the salt was dissolved in hot acetonitrile (225 mL),the material was treated activated acrbon, filtered, and placed in a 5°C. refrigerator overnight. The white solids were filtered after 18hours, washed with cold acetonitrile (10 mL), and dried under highvacuum at room temperature until the product weight was constant (24hours). The process recovered L-threonine-valproic acid ester,hydrochloride SPIC003 (6.82 g, 77.5% recovery) as a white solid.

¹H NMR (300 MHz, DMSO): δ=8.71 (br s, 3H), 5.28 (m, 1H), 4.16 (d, 1H,J=2.7 Hz), 2.33 (m, 1H), 1.56-1.40 (m, 2H), 1.37-1.27 (m, 5H), 1.21-1.13(m, 4H), 0.84 (t, 6H, J=6.6 Hz).

¹³C NMR (75 MHz, DMSO): δ=173.97, 168.19, 67.69, 55.42, 44.43, 33.95,33.78, 20.07, 19.95, 16.54, 13.94.

HPLC analysis:

98.88% purity; r.t.=4.864 min.; 70% DIUF water/30% acetonitrile; 1mL/min; 40C; Luna C18, 5 u column (serial #211739-42), 4.6×250 mm; 20 ulinjection; sample dissolved in mobile phase.

CHN Analysis:

calc.: C, 51.15; H, 8.59; N, 4.97. found: C, 51.29; H, 8.59; N, 4.98.

Melting point: 144° C.

The procedure for the synthesis of the L-serine, L-serine(O-nitroxyester), L-hydroxyproline, and L-tyrosine conjugates of valproic acid(2-propylpentanoic acid) is outlined in Synthetic Sequence section. Thecomplete procedure and analytical data is given in the ExperimentalSection. In general, valproic acid (2-8 g, in batches) was coupled withthe corresponding protected amino acids. The protected intermediateswere purified by column chromatography and the protective groups wereremoved. The final amides were purified by crystallization from ether. Aportion of the L-serine amide was nitrated at low temperature withslight excess of a standard nitrating solution to form the nitroxyester, which could be separated from starting material bycrystallization from toluene. The final products were dried to aconstant weight before analysis by NMR, HPLC, melting point, and CHNanalysis.

Synthetic Sequence:

Synthesis of the L-Serine, L-Tyrosine, and L-Hydroxyproline Conjugatesof Valproic Acid a) Et₃N; b) LiOH; c) EDC, DMAP; d) H₂, 10% Pd/c, HCl;e) 90% HNO₃, AcOH Experimental Section

The synthesis of SPI0027, SPI0028, SPI0029 and SPI0030 was conducted inbatches (3-10 g). Reagents mentioned in the experimental section werepurchased at the highest obtainable purity from Lancaster,Sigma-Aldrich, Acros, or Bachem, except for solvents, which werepurchased from either Fisher Scientific or Mallinkrodt.

1) SPI0027 Hydroxy-(2(S)-propyl-pentanoylamino)-acetic acid(L-serine-valproic acid amide)

A) Preparation of SPI002701 (Protected Intermediate):

A mixture of 2-propylpentanoic acid (valproic acid 16.0 g, 0.11 mole)and thionyl chloride (30 mL) were stirred at room temperature for 4hours. After 4 hours, the solution was concentrated under reducedpressure to prepare the acid chloride of valproic acid (17.8 g, 98.8%yield). A portion of the remaining colorless oil (11.0 g) was addeddrop-wise to an ice-cold mixture of L-serine methyl ester, hydrochloride(10.60 g, 0.068 mole) and triethylamine (30 mL) in anhydrousdichloromethane (100 mL). After the addition, the mixture stirred for 2hours under an argon atmosphere, while cooling with an ice/water batch.The ice bath was removed and the mixture stirred for 2 hours at roomtemperature. The solvent and excess triethylamine were removed underreduced pressure and a fresh aliquot of dichloromethane (100 mL) wasadded. The mixture was then extracted with water (50 mL), 5%hydrochloric acid (2×50 mL), saturated sodium bicarbonate solution (2×50mL), and DIUF water (50 mL). The remaining dichloromethane solution wasdried over sodium sulfate (10 g), filtered, and concentrated underreduced pressure. The remaining light yellow solid (17.22 g) waspurified by column chromatography on silica gel (350 g, 0.035-0.070 mm,6 nm pore diameter), eluting with hexanes/ethyl acetate (1:2). Afterconcentration of the product containing fractions under reduced pressureand drying under high vacuum until the weight was constant, theexperiment produced the methyl ester protected L-serine-valproate amideSPI002701 (9.45 g, 57% yield) as a white solid.

¹H NMR (300 MHz, CDCl₃): δ=6.55 (1H, d, J=7.2 Hz), 4.70 (1H, m),4.0-3.89 (2H, m), 3.78 (3H, s), 3.16 (1H, m), 2.17 (1H, m), 1.61 (2H,m), 1.46-1.23 (6H, m), 0.90 (6H, t, J=7.2 Hz).

¹³C NMR (75 MHz, CDCl₃): δ=176.81, 171.08, 63.78, 54.67, 52.90, 47.61,35.37, 21.01, 20.91, 14.38.

B) Deprotection of SPI002701:

The protected methyl ester protected L-serine-valproate amide SPI002701(9.40 g, 38.3 mmol) was dissolved in THF (100 mL) and water (50 mL)containing lithium hydroxide (1.40 g, 58.4 mmol) that was cooled in anice-water batch. After stirring for 3 hours while cooling in ice, 10%hydrochloric acid (50 mL) and the product was extracted withdichloromethane (2×100 mL). The dichloromethane fractions were combined,dried over sodium sulfate (10 g), filtered and concentrated underreduced pressure. The crude product (9.6 g, light yellow solid) wasdissolved in diethyl ether (40 mL) and stored overnight at −10° C. Afterfiltration, the remaining white solid was dried at room temperatureunder high vacuum until the product weight was constant (24 hours). Theexperiment produced L-serine-valproic amide SPI0027 (5.62 g, 63% yield)as a white solid.

¹H NMR (300 MHz, DMSO): δ=12.3 (br s, 1H), 7.91 (d, 1H, J=7.5 Hz), 4.90(br s, 1H), 4.28 (m, 1H), 3.63 (m, 2H), 2.82 (m, 1H), 1.42 (m, 2H), 1.21(m, 6H), 0.82 (t, 6H, J=7 Hz).

¹³C NMR (75 MHz, DMSO): δ=174.92, 171.95, 61.51, 54.42, 44.85, 35.03,34.88, 20.18, 20.03, 14.18, 14.13.

HPLC Analysis:

100% purity; r.t.=4.867 min.; 35% DIUF water (0.1% TFA)/65% methanol; 1mL/min; 39.8 C; Synergi Polar-RP 5 u column (serial #234257), 4.6×250mm; 20 ul injection; DAD1 A, Sig=210.4, Ref=550, 100.

CHN Analysis:

calc.(C10H19NO4): C, 57.12; H, 9.15; N, 6.06. found: C, 57.34; H, 9.22;N, 5.89.

Melting point: 65-67° C.

2) SPI0028 2-Propyl-pentanoic acid 4-(2(S)-amino-2-carboxy-ethyl)-phenylester, hydrochloride, (L-Tyrosine-valproic acid ester, hydrochloride)

A) Preparation of SPI002801 (Protected Intermediate):

A mixture of 2-propylpentanoic acid (valproic acid, 2.66 g, 18.4 mmole),N-carbobenzyloxy-L-tyrosine benzyl ester (5.0 g, 12.3 mmole), EDC (3.54g, 18.4 mmole), and DMAP (150 mg, 1.23 mmole) in anhydrousdichloromethane (25 mL) was stirred under an argon atmosphere at roomtemperature for 2 hours. After 2 hours, the dichloromethane solution waswashed with water (2×25 mL), 5% hydrochloric acid (2×25 mL), 5% sodiumbicarbonate (2×25 mL), and DIUF water (25 mL). The dichloromethane wasdried over magnesium sulfate, (5 g), filtered and concentrated underreduced pressure. The remaining white solid (8.14 g) was purified bycolumn chromatography on silica gel (120 g), eluting with 3:1heptane/ethyl acetate. The product containing fractions were combined,washed with additional saturated sodium bicarbonate (100 mL, to removevalproic acid), and dried over sodium sulfate (10 g). Afterconcentration and drying to a constant weight, the experiment generatedthe protected intermediate SPI002801 (6.5 g, 99% yield) as a whitesolid.

¹H NMR (300 MHz, CDCl₃): δ=7.33 (10H, m), 6.98 (2H, d, J=8.4 Hz), 6.89(2H, d, 8.4 Hz), 5.26-5.09 (4H, m), 4.68 (1H, dd, J=13.5, 6.6 Hz), 3.09(2H, m), 2.59 (1H, m), 1.80-1.36 (8H, m), 0.96 (6H, t, J=7.2 Hz).

¹³C NMR (75 MHz, CDCl₃): δ=174.86, 171.24, 155.64, 149.90, 135.06,133.01, 130.34, 128.71, 128.59, 128.24, 128.18, 121.76, 67.50, 67.18,54.99, 45.58, 45.22, 37.76, 34.92, 34.63, 20.97, 20.83, 14.33.

B) Deprotection of SPI002801:

The protected L-tyrosine-valproate ester SPI002801 (6.5 g) was dissolvedin ethyl acetate (100 mL) at room temperature and added to a Parr bottle(500 mL) that contained 10% palladium on carbon (2.25 g, 50% wet) undera nitrogen atmosphere. The nitrogen atmosphere was replaced withhydrogen gas (32 psi). After 5 hours of shaking, the ethyl acetate wasacidified with 2N hydrogen chloride in diethyl ether (20 mL) and thecatalyst was removed by filtration through a thin layer of celite 521(20 g). The ethyl acetate mixture was concentrated under reducedpressure at room temperature. After drying for 2 hours under high vacuumat room temperature, The remaining solid (5.4 g) was stirred in diethylether (100 mL) overnight under an argon atmosphere. After filtration anddrying to a constant weight under high vacuum, the experiment producedL-tyrosine-valproic acid ester SPI0028 (3.50 g, 72% yield) as a whitesolid.

¹H NMR (300 MHz, DMSO): δ=8.63 (br s, 3H), 7.34 (d, 2H, J=8.4 Hz), 7.02(d, 2H, J=8.4 Hz), 4.11 (br t, 1H), 3.18 (d, 2H, J=4.8 Hz), 2.57 (m,1H), 1.66-1.45 (m, 4H), 1.45-1.29 (m, 4H), 0.91 (t, 6H, J=7.5 Hz).

¹³C NMR (75 MHz, DMSO): δ=174.02, 169.99, 149.36, 132.52, 130.62,121.55, 53.16, 44.47, 34.95, 34.15, 20.19, 13.97.

HPLC Analysis:

99.75% purity; r.t.=7.433 min.; 60% DIUF water (0.1% TFA)/40%acetonitrile; 1 mL/min; 39 C; Luna C18, 5 u column (serial #191070-3),4.6×250 mm; 20 ul injection; DAD1 A, Sig=210, Ref=550, 100.

CHN Analysis:

calc.(C17H26ClNO4): C, 59.38; H, 7.62; N, 4.07. found: C, 59.46; H,7.58; N, 4.14.

Melting point: 192.0-194.0° C.

3) SPI00294(R)-Hydroxy-1-(2-propyl-pentanoyl)-pyrrolidine-2(5)-carboxylic acidValproic-L-hydroxyproline amide

A) Preparation of SPI002901 (protected intermediate):

A mixture of 2-propylpentanoic acid (valproic acid 2.95 g, 0.02 mole)and thionyl chloride (8 mL) were stirred at room temperature for 4hours. After 4 hours, the solution was concentrated under reducedpressure. The remaining colorless oil was added drop-wise to an ice-coldmixture of L-hydroxyproline benzyl ester, hydrochloride (Bachem, 6.2 g)and triethylamine (10 mL) in anhydrous dichloromethane (50 mL). Afterthe addition, the mixture stirred for 2 hours under an argon atmosphere,while cooling with an ice/water batch. The ice bath was removed and themixture stirred for 4 hours at room temperature. The solvent and excesstriethylamine were removed under reduced pressure and a fresh aliquot ofdichloromethane (100 mL) was added. The mixture was then extracted withwater (50 mL), 5% hydrochloric acid (2×50 mL), saturated sodiumbicarbonate solution (2×50 mL), and DIUF water (50 mL). The remainingdichloromethane solution was dried over sodium sulfate (10 g), filtered,and concentrated under reduced pressure. The remaining crude product(6.2 g) was purified by column chromatography on silica gel (110 g,0.035-0.070 mm, 6 nm pore diameter), eluting with hexanes/ethyl acetate(1:1). After concentration of the product containing fractions underreduced pressure and drying under high vacuum until the weight wasconstant, the experiment produced the protectedL-hydroxyproline-valproate amide SPI002901 (5.70 g, 80% yield) as acolorless oil.

¹H NMR (300 MHz, CDCl₃): δ=7.37-7.27 (5H, m), 5.14 (2H, d, J=12.6 Hz),4.63 (1H, t, J=7.8 Hz), 4.52 (1H, m), 3.76 (1H, dd, J=10.4, 4.2 Hz),3.58 (1H, dd, J=10.4, 1.5 Hz), 2.47 (1H, m), 2.25 (1H, m), 2.04 (1H, m),1.64-1.55 (2H, m), 1.43-1.19 (6H, m), 0.8 (6H, m).

¹³C NMR (75 MHz, CDCl₃): δ=175.96, 172.21, 135.67, 128.56, 128.23,70.15, 67.00, 58.02, 55.33, 43.56, 37.76, 35.44, 35.08, 21.02, 20.71,14.51.

The protected L-hydroxyproline-valproate ester SPI002901 (9.3 g, 26.7mmole) was dissolved in ethyl acetate (25 mL) at room temperature andadded to a Parr bottle (500 mL) that contained 10% palladium on carbon(1.53 g, 50% wet) under a nitrogen atmosphere. The nitrogen atmospherewas replaced with hydrogen gas (35 psi). After 20 hours the catalyst wasremoved by filtration through a thin layer of activated carbon, washingwith ethanol (25 mL). The ethanol and ethyl acetate were concentratedunder reduced pressure at room temperature. After drying under highvacuum, the remaining solid (6.54 g) was crystallized from t-butylmethyl ether (100 mL, 48 hours at −10° C.). After filtration and dryingto a constant weight, the experiment produced L-hydroxyproline-valproicamide SPI0029 (5.17 g, 83% yield) as a white solid.

¹H NMR (300 MHz, DMSO): δ=12.6 (br s, 1H), 5.39 (br s, 1H), 4.69-4.48(m, 2H), 3.90-3.58 (m, 4H), 2.78 (m, 1H), 2.51-2.32 (m, 2H), 2.22-2.12(m, 1H), 1.80-1.42 (m, 8 H), 1.11 (m, 6H).

¹³C NMR (75 MHz, DMSO): δ=173.80, 173.43, 68.78, 75.42, 55.04, 42.10,37.29, 35.13, 34.74, 20.17, 19.74, 14.28, 14.24.

HPLC Analysis:

99.89% purity; r.t.=7.20 min.; 70% DIUF water (0.1% TFA)/30% ACN; 1mL/min; 39.8 C; Synergi Polar-RP 5 u column (serial #161309), 4.6×250mm; 20 ul injection; DAD1 A, Sig=210.4, Ref=550, 100.

Specific Rotation: −56.8 (5 mg/mL in ethanol at 25 C)

CHN Analysis:

calc.(C13H23NO4): C, 60.68; H, 9.01; N, 5.44. found: C, 60.71; H, 9.03;N, 5.45.

Melting point: 113-114° C.

4) SPI0030 3-Nitroxy-2(S)-(2-propyl-pentanoylamino)-propionic acid(L-serine(O-nitroxy ester)-valproic acid amide)

The L-serine valproic amide (SPI0029, 8.4 g, 0.036 mole) was dissolvedin glacial acetic acid (60 mL). The flask was cooled in an ice/waterbath under an argon atmosphere and cold 90% nitric acid (6.0 mL) wasadded. After 4 hours at 5° C., the solution was poured into ice (400 g)and extracted with dichloromethane (2×150 mL). The dichloromethanefractions were combined, dried over sodium sulfate (15 g), filtered andconcentrated. Toluene (150 mL) was added to the remaining oil and thesolution was concentrated a second time to remove acetic acid. Afterdrying under high vacuum to a constant weight, the remaining oil (12.4g) was dissolved in toluene (50 mL) and stored at −10° C. overnight.After 72 hours at −10° C., the solids were filtered and dried to aconstant weight. The experiment produced the nitroxy ester of theL-serine-valproic amide SPI0030 (4.37 g, 43% yield) as a white solid.

¹H NMR (300 MHz, CDCl₃): δ=8.88 (br s, 1H), 6.60 (d, 1H, J=7.2 Hz), 5.00(m, 1H), 4.87 (m, 2H), 2.23 (m, 1H), 1.56 (m, 2H), 1.43 (m, 2H), 1.29(m, 6H), 0.89 (t, 6H, J=7.2 Hz).

¹³C NMR (75 MHz, CDCl): δ=177.83, 170.88, 71.42, 50.60, 47.47, 35.15,20.88, 14.28.

HPLC Analysis:

99.25% purity; r.t.=5.400 min.; 65% methanol/35% phosphate buffer(pH=3); 1 mL/min; 36.0 C; Synergi Polar-RP 5 u column (serial#161309-2), 4.6×250 mm; 20 ul injection; Sig=210.4.

Specific Rotation: −19.7 (10.1 mg/mL in ethanol at 25 C)

CHN Analysis:

calc.(C11H20N2O6): C, 47.82; H, 7.30; N, 10.14. found: C, 48.12; H,7.19; N, 9.84.

Melting point: 49.0-51.0° C.

Solubility of the above esters were determined in water at roomtemperature by dissolving excess of each of the drug and allowing themto settle for a few hours. The resulting solutions were centrifuged at1500 rpm for 3 min and the supernatant liquid was analyzed. It was shownthat these esters possess solubility in water in excess of 50 mg/mL.

There are a number of screening tests to determine the utility of thederivatives created according to the disclosed methods. These includeboth in vitro and in vivo screening methods.

The in vitro methods include acid/base hydrolysis of the derivatives,hydrolysis in pig pancreas, hydrolysis in rat intestinal fluid,hydrolysis in human gastric fluid, hydrolysis in human intestinal fluid,and hydrolysis in human blood plasma. These assays are described inSimmons, D M, Chandran, V R and Portmann, G A, Danazol, Amino AcidDerivatives: In Vitro and In Situ Biopharmaceutical Evaluation, DrugDevelopment and Industrial Pharmacy, Vol. 21, Issue 6, Page 687, 1995,the contents of all of which are incorporated by reference.

The amino acid derivatives of Valproic acid of the present invention areeffective in treating diseases or conditions in which Valproic acidnormally is used. Without wishing to be bound, it is believed that theamino acid derivatives disclosed herein are transformed within the bodyto release the active compound. Alternatively, the amino acid derivativemay not be transformed to release the active component in the body. Thislatter form enhances the therapeutic benefits of the Valproic acid byreducing or eliminating biopharmaceutical and pharmacokinetic barriersassociated with each of them. However it should be noted that thesederivatives themselves will have sufficient activity without releasingany active drug in the mammals.

Thus, the amino acid derivative of the present invention enhances thetherapeutic benefits by removing biopharmaceutical and pharmacokeneticbarriers of existing drugs. Furthermore, the derivatives are easilysynthesized in high yields using reagents which are readily andcommercially available.

Synthesis of Clopidogrel Derivatives Overview:

The procedure for the synthesis of the L-serine and L-threonine estersof clopidogrel is outlined in Synthetic Sequence section. The completeprocedure and analytical data are given in the Experimental Section. Ingeneral, the hydrosulfate salt of clopidogrel (Xiangding ChemicalInternational Company) was treated with sodium bicarbonate to generatethe free amine. The methyl ester was then removed by displacement withlithium iodide in pyridine. The unwanted isomer generated in thereaction was partially removed by crystallization from water. The acidintermediate was then coupled with the tert-butyl ester of boc-L-serineor boc-L-threonine using EDC. The protected intermediate esters werepurified twice by column chromatography and treated with dilutehydrochloric acid in acetic acid at low temperature to remove theprotective groups. Washing the final salts with ethyl acetate purifiedthe amino acid ester salts of clopidogrel. The final salts were driedunder high vacuum and shipped to Signature Pharmaceuticals Inc., afteranalysis by NMR, HPLC, CHN, specific rotation, and melting point.

Synthetic Sequence:

Synthesis of the L-Serine and L-Threonine Esters of Clopidogrel a)NaHCO₃; b) LiI, pyridine; c) boc-THR-(OtBu), EDC, DMAP; d)boc-SER-(OtBu), EDC, DMAP; e) 1M HCl, AcOH Experimental Section

The syntheses of SPIB00301 and SPIB00302 were conducted in batches.Reagents mentioned in the experimental section were purchased at thehighest obtainable purity from Lancaster, Sigma-Aldrich, Acros, Bachem,or Xiangding Chemical International, except for solvents, which werepurchased from either Fisher Scientific or Mallinkrodt.

1) Preparation of Clopidogrel Acid

To a solution of clopidogrel hydrosulfate (97.7 g, 233 mmol) in DIUFwater (1 L) was added sodium bicarbonate (39.1 g, 466 mmol) in smallportions. After mixing, t-butyl methyl ether (1 L) was added and thesolution stirred for 30 minutes. The layers were separated and theaqueous layer was extracted a second time with t-butyl methyl ether (300mL). The organic layers were combined, washed with brine (500 mL), anddried over sodium sulfate. After filtration, the t-butyl methyl etherwas removed under reduced pressure. The remaining clopidogrel (yellowoil, 77.82 g, 104% yield) was dried under high vacuum at roomtemperature for 18 hours until most of the t-butyl methyl ether wasremoved. The clopidogrel (77.8 g, 242 mmol) was dissolved in pyridine(200 mL) and added to a refluxing solution of anhydrous lithium iodide(120 g, 897 mmol) in pyridine (800 mL). After 8 hours at reflux, theflask was cooled to room temperature and the pyridine was removed underreduced pressure (at 40° C.). The remaining wet solid (470 g) wasdissolved in DIUF water (300 mL) and acidified to pH=5.5 with aceticacid (50 mL). The product was then extracted with dichloromethane (3×300mL). The dichloromethane fractions were combined, dried over sodiumsulfate, filtered, concentrated under reduced pressure, and dried underhigh vacuum. The remaining yellow mixture (161.3 g) was mixed withanhydrous THF (500 mL) for 60 minutes. After 60 minutes, the salts wereremoved by filtration. The filtrate was concentrated under reducedpressure to leave a brown oil (128.3 g), which was dissolved in DIUFwater (3×200 mL) and freeze-dried under high vacuum to remove traces ofsolvent. The remaining yellow solid (108.5 g) was again added to water(600 mL) and sonicated. The solids were removed by filtration and thefiltrate was freeze-dried under high vacuum. The remaining solid (68.7g) was partitioned between water (400 mL) and t-butyl methyl ether (400mL), stirred for 24 hours, stored over the weekend at 5° C., andfiltered. The layers were separated and the aqueous layer was extracteda second time with t-butyl methyl ether (300 mL). The purified productwas then extracted from the aqueous layer with dichloromethane (2×200mL). The dichloromethane fractions were combined, dried over sodiumsulfate, filtered, concentrated and freeze-dried (from 100 mL DIUFwater). The procedure generated the acid of clopidogrel (28.5 g) as ayellow solid.

¹H NMR (300 MHz, CDCl₃): δ 8.02 (1H, s br), 7.88 (1H, d, J=7.5 Hz), 7.37(1H, d, J=8.1 Hz), 7.22 (1H, t, J=7.5 Hz), 7.13 (2H, m), 6.65 (1H, d,J=4.8 Hz), 5.14 (1H, s), 4.14 (2H, m), 3.37 (1H, m), 3.28 (1H, m), 3.00(2H, m).

¹³C NMR (75 MHz, CDCl₃): δ 169.75, 135.08, 131.71, 131.49, 130.07,129.98, 129.82, 129.08, 127.44, 125.06, 124.14, 67.23, 50.22, 48.30,22.62.

Specific rotation: +43.5 deg (23° C., 0.0216 g/2 mL methanol, 589 nm)

2) Preparation of the L-Threonine Ester of Clopidogrel CouplingProcedure:

A mixture of the acid of clopidogrel (14.1 g, 45.8 mmol),boc-L-threonine tert-butyl ester (11.4 g, 41.6 mmol, prepared by theliterature method), 1-[3-dimethylamino)propyl]-3-ethylcarbodiimidehydrochloride (EDCI, 7.38 g, 38.5 mmol), and DMAP (0.783 g, 6.4 mmol) inanhydrous dichloromethane (300 mL) was stirred at room temperature underan argon atmosphere for 22 hours. Additional dichloromethane (1.2 L) wasadded and the dichloromethane solution was washed with water (1200 mL)and 5% sodium bicarbonate solution (1200 mL). After drying thedichloromethane solution over sodium sulfate, filtration, andconcentration under reduced pressure, the residue (24 g) was purified bycolumn chromatography on silica gel (500 g), eluting with heptane-ethylacetate (8:1). The product containing fractions were combined andconcentrated to provide the protected L-threonine ester of clopidogrelSPIB0030101 (13.5 g, 52% yield), as clear liquid.

¹H NMR (300 MHz, CDCl₃): δ 7.69 (1H, m), 7.42 (1H, dd, J=7.2, 2.1 Hz),7.29 (2H, m), 7.06 (1H, d, J=5.1 Hz), 6.65 (1H, d, J=5.1 Hz), 5.47 (1H,m), 5.16 (1H, m), 4.84 (1H, s), 4.30 (1H, d, J=3.6 Hz), 3.65 (2H, m),2.85 (4H, m), 1.46 (18H, m), 1.15 (3H, d, J=6 Hz).

¹³C NMR (75 MHz, CDCl₃): δ 169.46, 168.91, 155.89, 134.63, 133.68,133.23, 129.96, 129.85, 129.52, 127.27, 125.22, 122.80, 82.61, 80.14,71.62, 68.16, 57.65, 50.72, 48.51, 28.50, 27.89, 25.65, 16.84.

Deprotection and Purification:

A solution of hydrogen chloride (1.0 M in acetic acid, 210.6 mL) wasadded drop-wise to a solution of the protected L-threonine ester ofclopidogrel SPIB0030101 (11.9 g, 21.06 mmol) in anhydrousdichloromethane (115 mL) that was cooled in an ice batch, under an argonatmosphere. The mixture was stored for 6.5 days at 5° C. The mixture wasconcentrated under reduced pressure and dried under high vacuum togenerate a light, yellow solid (13.22 g). Ethyl acetate (150 mL) wasadded to the solid and the mixture was sonicated for 5 minutes. Theethyl acetate was removed by filtration and the remaining solid wasadded to an additional volume of ethyl acetate (150 mL) and the mixturewas sonicated a second time for 5 minutes. After filtration thesonication process was repeated a third time. The white, solid product(10.5 g) was filtered and dissolved in dichloromethane (320 mL). Ethylacetate (150 mL) was added and the solution was stored at −20° C.overnight. The solids were filtered and the crystallization process wasrepeated with methanol (1.5 mL)/dichloromethane (180 mL) and ethylacetate. The solids were filtered and freeze-dried under high vacuum.The procedure generated the clopidogrel-L-threonine ester,dihydrochloride SPIB00301 (6.1 g, 60% yield, 99.51% purity by HPLC) inmonohydrate form (as white solid).

¹H NMR (300 MHz, DMSO): δ 7.78-7.58 (m, 4H), 7.42 (m, 1H), 6.80 (m, 1H),5.95 (m, 1H), 5.85 (m, 1H), 4.43 (m, 1H), 4.31-4.17 (m, 3H), 3.84 (m,2H), 3.33 (m, 2H), 1.33 (d, 3H, J=6 Hz).

¹³C NMR (75 MHz, DMSO): δ 170.56, 167.11, 136.33, 134.39, 132.46,129.93, 127.46, 126.66, 126.51, 126.01, 73.09, 66.88, 62.91, 57.94,51.81, 22.93, 16.75.

HPLC Analysis:

99.51% purity, r.t.=10.042 min, sample dissolved in DIUF water/ACN, 75%DIUF water (0.1% TFA)/25% ACN, Hydro-RP (#162383-7), 4 u, 250×4.6 mm, 1mL/min., 40° C., 20 uL inj. vol., SPD-10Avp, chl-210 nm.

Melting point: 110-112° C. (Thomas Hoover CMPA, uncorrected)

Specific rotation: +22.5 deg (25° C., 27.7 mg/5 mL ethanol 589 nm)

CHN Analysis:

calc.: C, 45.66; H, 5.04; N, 5.60, Cl, 21.28 (C₁₉H₂₅Cl₃N₂O₅S). found: C,44.89, H, 4.96, N, 5.61, C121.32.

3) Preparation of the L-Serine Ester of Clopidogrel Coupling Procedure:

A mixture of the acid of clopidogrel (10.24 g, 33.2 mmol), boc-L-serinetert-butyl ester (7.80 g, 29.8 mmol),1-[3-dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDCI, 8.94g, 46.6 mmol), and DMAP (0.25 g) in anhydrous dichloromethane (200 mL)was stirred at room temperature under an argon atmosphere for 4 hours.The dichloromethane solution was washed with water (2×200 mL) andsaturated sodium bicarbonate (250 mL). After drying over sodium sulfate,filtration, and concentration under reduced pressure, the remainingyellow oil (18.62 g) was purified twice by column chromatography onsilica gel (300 g), on it eluting with heptane-ethyl acetate (9:1). Theproduct containing fractions were combined in two fractions andconcentrated. The procedure generated the protected L-serine ester ofclopidogrel SPIB0030201 (6.32 g, 34% yield).

¹H NMR (300 MHz, CDCl₃): δ 7.67 (1H, m), 7.42 (1H, m), 7.29 (2H, m),7.06 (1H, d, J=5.1 Hz), 6.66 (1H, d, J=5.1 Hz), 4.95 (1H, d, J=7.8 Hz),4.89 (1H, s), 4.44 (3H, d), 3.72 (1H, d, J=14.4 Hz), 3.60 (1H, d, J=14.4Hz), 2.87 (4H, m), 1.42 (18H, m).

¹³C NMR (75 MHz, CDCl₃): δ 170.16, 168.19, 154.79, 134.48, 133.61,133.11, 129.81, 129.75, 129.42, 127.13, 125.10, 122.64, 82.72, 79.94,71.62, 67.77, 64.71, 53.46, 50.66, 48.39, 28.36, 27.97, 25.55.

Deprotection and Purification:

A solution of hydrogen chloride (1.0 M in acetic acid, 100 mL) was addeddrop-wise to a solution of the protected L-serine ester of clopidogrelSPIB0030201 (6.30 g, 11.43 mmol) in anhydrous dichloromethane (100 mL)that was cooled in an ice batch, under an argon atmosphere. The mixturewas stored for 4 days at 5° C. The mixture was concentrated underreduced pressure and dried under high vacuum to generate a colorlessgel. Ethyl acetate (250 mL) was added to the gel and the mixture wassonicated for 5 minutes. The white, solid product was filtered and driedunder high vacuum. The white, solid product (6.4 g) was filtered anddissolved in dichloromethane (250 mL). Ethyl acetate (150 mL) was addedand the solution was stored at −20° C. overnight. After filtration andfreeze-drying, the procedure generated the clopidogrel-L-serine ester,dihydrochloride SPIB00302 (4.8 g, 97% yield, 96.64% purity by HPLC) inmonohydrate form (as off-white solid).

¹H NMR (300 MHz, DMSO): δ 9.18 (br s, 3H), 8.16 (d, 1H, J=6.6 Hz),7.66-7.40 (m, 4H), 6.87 (m, 1H), 5.76 (br s, 1H), 4.75 (m, 2H), 4.49 (m,1H), 4.40-4.0 (m, 1H). 3.40-3.80 (m, 1H), 3.17 (m, 2H).

¹³C NMR (75 MHz, DMSO): δ 167.50, 164.29, 134.23, 132.29, 131.35,130.93, 130.41, 128.26, 127.81, 126.98, 125.33, 124.73, 66.12, 63.95,50.82, 50.15, 49.12, 22.13.

HPLC Analysis:

96.64% purity, r.t.=10.56 min, sample dissolved in DIUF water/ACN, 83%DIUF water (0.1% TFA), 17% ACN, Gemini-C18 (#262049-2), 5 u, 250×4.6 mm,1 mL/min., 40° C., 20 uL inj. vol., SPD-10Avp, chl-210 nm.

Specific rotation: +42.0 deg (25° C., 34.0 mg/5 mL ethanol 589 nm)

Melting point: 100-102° C. (Thomas Hoover CMPA, uncorrected)

CHN Analysis:

calc.: C, 44.50; H, 4.77; N, 5.77, Cl, 21.89 (C₁₈H₂₃Cl₃N₂O₅S). found: C,44.63, H, 4.69, N, 5.76, Cl, 22.20.

Synthesis of Danazol Proline Ester Overview:

The procedure for the synthesis of the L-proline esters of Danazol isoutlined in Synthetic Sequence section. The complete procedure andanalytical data is given in the Experimental Section. In general,Danazol (4-7 g, in batches) was coupled withN-tert-butoxycarbonyl-L-proline(1.2-1.6 equivalents) with1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, hydrochloride (EDC,1.2-1.4 equivalents) in the presence of catalytic amount of4-(N,N-dimethylamino)-pyridine (DMAP). Once the reaction was complete,the excess EDC and N-tert-butoxycarbonyl-L-proline were removed byextraction. The crude protected ester of Danazol was purified by columnchromatography on silica gel. Unreacted Danazol was collected andreprocessed. The yield for the coupling step was 80-96%. The protectivegroup was removed with 2M hydrogen chloride solution in diethyl ether atlow temperature. The hydrochloride salt of the Danazol-L-proline esterwas purified as the free base by column chromatography. Once pure, thefree base of Danazol-L-proline ester was converted to the hydrochloridesalt with diluted hydrogen chloride solution in diethyl ether, at lowtemperature.

Synthetic Sequence:

Synthesis of the L-Proline Ester of Danazol a) EDC, DMAP, CH₂Cl₂; b)HCl, diethyl ether; c) NaHCO3, CH₂Cl₂; d) HCl, CH₂Cl₂, diethyl etherExperimental Section

The synthesis of SPIC004 was conducted in several small batches. Arepresentative batch for each step is described below. Reagentsmentioned in the experimental section were purchased at the highestobtainable purity from Spectrum, Fluka, Acros and Sigma-Aldrich.

1) Synthesis of SPC00401 Danazol-N-tert-butoxycarbonyl-L-proline ester

Danazol (7.0 g, 20.74 mmole), N-tert-butoxycarbonyl-L-proline (4:55 g,21.14 mmole), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide,hydrochloride (EDC, 4.0 g, 20.86 mmole) and4-(N,N-dimethylamino)-pyridine (DMAP, 0.27 g, 2.2 mmole) were dissolvedin anhydrous dichloromethane (28 ml) while stirring and cooling in anice-water bath. The ice bath was removed and the mixture was allowed tostir at room temperature for 24 hours. After stirring under an argonatmosphere at room temperature for 24 hours, additionalN-tert-butoxycarbonyl-L-proline (0.9 g, 4.18 mmole), EDC (0.8 g, 4.17mmole) and DMAP (0.06 g, 0.5 mmole) were added to the reaction mixture.After 6 days of stirring under an argon atmosphere at room temperature,additional reagents were added: N-tert-butoxycarbonyl-L-proline (0.9 g,4.18 mmole), EDC (0.8 g, 4.17 mmole) and DMAP (0.06 g, 0.5 mmole). After8 days, the reaction mixture was diluted with dichloromethane (40 mL),and extracted with saturated sodium bicarbonate solution (2×100 mL) andDIUF water (3×100 mL). After drying over anhydrous sodium sulfate, thesolution was filtered and concentrated under reduced pressure. The lightyellow solid foam (12.6 g) that remained was purified by columnchromatography on silica gel (450 g, 0.035-0.070 mm, 6 nm porediameter), eluting with toluene:acetone (9:1). The product containingfractions were combined, concentrated under reduced pressure, and driedunder high vacuum until a constant weight was obtained. The experimentproduced the protected Danazol-L-proline ester (SPIC00401) as a white,solid foam (7.8 g, 70.3% yield). A second fraction containing a mixtureof Danazol and the protected Danazol-L-proline ester (3.7 g) was alsorecovered. The recovered mixture was reprocessed (as above) to producean additional amount of SPIC00401 (2.88 g, 26.0%).

SPIC00401 Pyrrolidine-1,(2S)-dicarboxylic acid 1-tert-butyl ester2-(1-ethynyl-10a,12a-dimethyl-2,3,3a,3b,4,5,10,10a,10b,11,12,12a-dodecahydro-1H-7-oxa-8-aza-dicyclopentala[a,h]phenanthren-1-yl)ester

¹H NMR (300 MHz, CDCl₃): δ=8.00 (1H, s), 6.17 (1H, s), 4.32-4.20 (1H,m), 3.58-3.35 (2H, m), 2.84-1.0 (37H, m). (Mixture of conformationalisomers)

¹³C NMR (75 MHz, CDCl₃): δ=171.52, 171.21, 164.82, 154.27, 154.09,153.77, 148.68, 108.94, 107.68, 85.06, 84.85, 82.94, 80.07, 79.71,75.57, 75.15, 59.40, 58.92, 53.87, 48.47, 48.36, 47.69, 46.66, 46.45,41.18, 37.51, 36.84, 33.54, 33.08, 32.38, 31.07, 30.89, 29.77, 28.69,28.57, 24.39, 23.95, 23.72, 21.72, 18.97, 13.77. (Mixture ofconformational isomers)

2) Synthesis of SPIC004 Danazol-L-proline ester, hydrochloride

To a stirred solution of protected Danazol-L-proline ester SPIC00401(7.8 g, 14.58 mmole) in anhydrous diethyl ether (32 mL) cooled in anice-water bath, a solution of hydrogen chloride in diethyl ether (73 mL,2M, 146 mmole) was added drop-wise under an argon atmosphere. After 4days stirring at room temperature, the mixture was concentrated underreduced pressure and dried under high vacuum. To the remaining yellowsolid (6.68 g) was added anhydrous diethyl ether (70 mL). The mixturewas stirred at room temperature for 20 hours under an argon atmosphere.Crude Danazol-L-proline ester, hydrochloride SPIC004 (4.6 g, 67.0%yield, HPLC purity 94%) was isolated as a light, yellow solid.

3) Conversion and purification of SPIC004b: Danazol-L-proline ester

To a cold solution of Danazol-L-proline ester, hydrochloride SPIC004(10.45 g, 22.18 mmole) in DIUF water (100 mL) was added dichloromethane(100 mL). Solid sodium bicarbonate (3.72 g, 44.36 mmole) was added inportions. The layers were separated and the aqueous layer was extractedwith dichloromethane (2×50 mL). The organic layer was dried overanhydrous sodium sulfate, filtered, and concentrated under reducedpressure. The remaining oil (12.1 g) was purified by columnchromatography on silica gel, eluting with 2-propanol. The productcontaining fractions were combined and concentrated under reducedpressure. The remaining yellow solid foam (8.44 g) was dissolved at roomtemperature in anhydrous diethyl ether (85 mL) and kept at −10° C. for24 hours. After filtration of the solid product and drying under highvacuum, Danazol-L-proline ester SPIC004b (5.89 g, 61.0% yield) as alight yellow solid (powder) was isolated.

Pyrrolidine-(2S)-carboxylic acid1-ethynyl-10a,12a-dimethyl-2,3,3a,3b,4,5,10,10a,10b,11,12,12a-dodecahydro-1H-7-oxa-8-aza-dicyclopenta[a,h]phenanthren-1-yl ester

¹H NMR (300 MHz, CDCl₃): δ=8.00 (1H, s), 6.18 (1H, s), 3.73 (1H, m),3.10 (1H, m), 2.89 (1H, m), 2.76-2.70 (2H, m), 2.62-2.35 (3H, m),2.25-1.07 (18H, m), 1.02 (3H, s), 0.91 (3H, s).

¹³C NMR (75 MHz, CDCl₃): δ=173.93, 164.83, 154.16, 148.64, 108.91,107.66, 85.09, 82.97, 75.30, 59.94, 53.85, 48.49, 47.73, 47.17, 41.17,37.53, 36.85, 33.54, 33.26, 32.37, 30.88, 30.31, 25.63, 23.91, 21.38,18.98, 13.67.

4) Regeneration of SPIC004 Danazol-L-proline ester, hydrochloride

Hydrogen chloride solution (26.8 mL, 1M, 26.8 mmole) was addeddrop-wise, under an argon atmosphere, to a stirred solution ofDanazol-L-proline ester SPIC004b (5.8 g, 13.35 mmole) in anhydrousdichloromethane (30 mL), cooled at −15° C. After stirring for 4 hours inan ice-water bath, the suspension was filtered, the solid dried underhigh vacuum at room temperature until the weight was constant. Theprocedure generated the purified Danazol-L-proline ester, hydrochlorideSPIC004 as a very light yellow solid (5.62 g, 89.3%, HPLC purity 98.2%).

Pyrrolidine-(2S)-carboxylic acid1-ethynyl-10a,12a-dimethyl-2,3,3a,3b,4,5,10,10a,10b,11,12,12a-dodecahydro-1H-7-oxa-8-aza-dicyclopenta[a,h]phenanthren-1-ylester, hydrochloride

¹H NMR (300 MHz, DMSO-d₆): δ=10.40 (1H, br s), 9.17 (1H, br s), 8.34(1H, s), 6.28 (1H, s), 4.36 (1H, dd, J=7.2, 6.9 Hz), 3.76 (1H, s), 3.22(2H, m), 2.73 (1H, d, J=15.9 Hz), 2.60-2.32 (7H, m), 2.0-1.35 (12H, m),1.13 (1H, m), 0.97 (3H, s), 0.93 (3H, s).

¹³C NMR (75 MHz, DMSO-d₆): δ=166.79, 163.73, 154.27, 148.86, 108.06,107.49, 85.95, 82.09, 78.68, 58.55, 53.15, 47.97, 47.26, 45.11, 40.44,36.41, 35.86, 32.69, 32.52, 31.35, 30.33, 27.73, 23.23, 22.89, 20.63,18.49, 13.26.

HPLC Analysis: 98.27% purity, r.t.=14.48 min, sample dissolved in DIUFwater/ACN, 50% DIUF water (0.1% TFA)/50% ACN, Gemini C18 (#262049-2), 5u, 250×4.6 mm, 1 mL/min., 40° C., 20 uL inj. vol., SPD-10Avp, chl-210nm.

CHN Analysis: calculated: C, 68.85; H, 7.49; N, 5.95, Cl, 7.53. found:C, 68.66; H, 7.49; N, 5.89, Cl, 7.76.

Specific rotation: −6.67 deg (20° C., 19.5 mg/l mL ethanol, 589 nm)

Melting point: 195.0-198.0° C. (dec.)

Preparation of Benazepril Esters

Overview:

The procedure for the synthesis of the L-serine, L-threonine, andL-hydroxyproline esters of benazepril is outlined in Synthetic Sequencesection. The complete procedure and analytical data are given in theExperimental Section. In general, benazepril (Xiangding ChemicalInternational Company) was butylated to protect the free acid group. Theethyl ester was then hydrolyzed to the acid with sodium hydroxide. Theamine present in the molecule was protected with a tert-butyloxycarbonylgroup to prevent dimerization during the subsequent coupling reaction.The protected intermediate was then coupled with the tert-butyl ester ofboc-L-serine, boc-L-threonine, or boc-L-hydroxyproline using EDC. Theprotected intermediate esters were treated with dilute hydrochloric acidin acetic acid at low temperature to remove all of the protectinggroups. The final amino acid ester salts of benazepril were purified bywashing with ethyl acetate and dichloromethane, dried under high vacuum,and analyzed by NMR, HPLC, CHN, specific rotation, and melting point.

Synthetic Sequence:

Synthesis of the L-Serine, L-Threonine, and L-Hydroxyproline Esters ofBenazepril a) THF; b) NaOH, MeOH, H₂O; c) (Boc)₂O, Et₃N, CH₂Cl₂; d)Boc-Ser-OtBu, EDC, DMAP, CH₂Cl₂; e) Boc-Thr-OtBu, EDC, DMAP, CH₂Cl₂; f)Boc-Hyp-OtBu, EDC, DMAP, CH₂Cl₂; g) HCl, AcOH, CH₂Cl₂ ExperimentalSection

The synthesis of SPIB00501, SPIB00502 and SPIB00503 was conducted inbatches. Reagents mentioned in the experimental section were purchasedat the highest obtainable purity from Lancaster, Sigma-Aldrich, Acros,Bachem, or Xiangding Chemical International, except for solvents, whichwere purchased from either Fisher Scientific or Mallinkrodt.

1) Butylation of Benazepril

A solution of 2-tert-butyl-1,3-dicyclohexyl isourea 1 (569 g, 2.03 mol,prepared by the literature procedure) in THF (500 mL) was addeddrop-wise to a mixture of benazepril hydrochloride (117 g, 0.254 mol) inanhydrous THF (500 mL) and cooled in an ice-water bath. After 2 hours,the ice bath was removed and the mixture was allowed to stir for 4 daysat room temperature under an argon atmosphere. The reaction mixture wasfiltered and the precipitate (DCU) was washed with methyl t-butyl ether(3×500 mL). The filtrates were combined and washed with 5% sodiumbicarbonate solution (1 L) and brine (1 L). The ether-THF solution wasdried over sodium sulfate, filtered, and concentrated under reducedpressure. The remaining mixture (202 g) was purified by columnchromatography on silica gel (1 Kg), eluting with heptane-ethyl acetate(4:1). The product containing fractions were combined and concentratedunder reduced pressure and dried at high vacuum till a constant weightwas achieved and a butylated benazepril 2 (88.17 g, 72% yield) wasisolated as a yellow oil.

¹H NMR (300 MHz, CDCl₃): δ 7.18 (9H, m), 4.61 (1H, d, J=16.8 Hz), 4.31(1H, d, J=16.8 Hz), 4.04 (2H, m), 3.25 (3H, m), 2.69 (2H, m), 2.57 (1H,m), 2.39 (1H, m), 2.19 (1H, s, br), 1.99 (3H, m), 1.43 (9H, s), 1.11(3H, t, J=7.2 Hz).

¹³C NMR (75 MHz, CDCl₃): δ 173.89, 173.58, 167.59, 141.21, 140.83,135.91, 129.27, 128.21, 128.16, 127.50, 126.53, 125.71, 122.04, 81.88,60.48, 60.18, 56.90, 51.16, 37.81, 35.13, 32.10, 28.52, 28.01, 14.14.

2) Saponification of the Butylated Benazepril

A sodium hydroxide solution (8.08 g, 0.202 mol) in DIUF water (540 mL)was added in a drop-wise fashion to the butylated benazepril 2 (88.17 g,0.184 mol) dissolved in methanol (1.6 L), while cooling the methanolsolution in ice bath. The solution was allowed to stir for 18 hoursunder an argon atmosphere while cooling in an ice bath. The reactionmixture was allowed to warm to room temperature and stir at roomtemperature for an additional 24 hours. The reaction mixture wasconcentrated under reduced pressure and dried under high vacuum toproduce a mixture of solid and liquid (130 g). The residue was cooled inan ice bath and a solution of acetic acid in water (1:10 v/v aceticacid/water) was added drop-wise to adjust the solution to pH=7. A whiteprecipitate formed which was filtered overnight in two crops (141.3 gand 7.05 g). The combined precipitates fractions were stirred withdichloromethane (500 mL) for one hour. After filtration and drying underhigh vacuum, 3 (48.6 g, 59% yield) was isolated as a white solid.

¹H NMR (300 MHz, CD₃OD): δ 7.34-7.16 (9H, m), 4.65 (1H, d, J=17.4 Hz),4.44 (1H, d, J=17.4 Hz), 3.87 (1H, dd, J=11.4, 7.8 Hz), 3.45 (1H, t,J=5.7 Hz), 3.37 (1H, dd, J=12.9, 5.4 Hz), 2.74 (3H, m), 2.60 (1H, m),2.34 (1H, m), 2.11 (2H, m), 1.43 (9H, s).

¹³C NMR (75 MHz, CD₃OD): δ 172.67, 168.85, 168.71, 142.04, 140.99,135.75, 130.65, 129.51, 129.38, 129.24, 128.69, 127.05, 123.88, 83.38,63.51, 59.07, 52.20, 34.95, 34.29, 32.47, 28.40, 28.27.

3) Amine Protection

A solution of di-tert-butyl dicarbonate (38.2 g, 175 mmol) in anhydrousdichloromethane (200 mL) was added in a drop-wise fashion to a solutionof acid 3 (72.0 g, 159 mmol) and triethylamine (64.4 g, 636 mmol) inanhydrous dichloromethane (600 mL) cooled in an ice bath, under an argonatmosphere. The mixture was allowed to stir at room temperature for 3days under an argon atmosphere. The reaction mixture was extracted withice-cold 0.5N hydrochloric acid (1.3 L) and brine (600 mL). Thedichloromethane solution was dried over sodium sulfate, filtered, andconcentrated under reduced pressure. The remaining yellow foam (87.1 g)was purified by column chromatography on silica gel (1 Kg), eluting withheptane-ethyl acetate (4:1). The product containing fractions werecombined, concentrated under reduced pressure, and dried under highvacuum. The amine 4 (40.0 g, 45% yield) was isolated as a white solid.

¹H NMR (300 MHz, CDCl₃): δ 13.22 (1H, s, br), 7.32-7.19 (9H, m), 4.80(1H, dd, J=12.3, 7.8 Hz), 4.75 (1H, d, J=16.8 Hz), 4.17 (1H, d, J=16.8Hz), 3.75 (1H, dd, J=9.0, 3.3 Hz), 3.18 (1H, m), 2.86 (2H, m), 2.66 (1H,m), 2.53 (1H, m), 2.00 (1H, m), 1.78 (1H, m), 1.60 (1H, m), 1.41 (9H,s), 1.40 (9H, s).

¹³C NMR (75 MHz, CDCl₃): δ 175.15, 171.44, 166.64, 154.36, 141.25,139.77, 134.87, 129.28, 128.69, 128.41, 128.26, 127.55, 126.03, 122.54,82.94, 82.59, 57.07, 55.39, 51.76, 34.73, 33.42, 32.95, 28.07, 28.00,27.85.

4) Preparation of the L-Serine Ester of Benazepril Coupling Procedure:

A mixture of acid 4 (15.5 g, 28.0 mmol), boc-L-serine tert-butyl ester(6.66 g, 25.5 mmol, prepared by the literature method),1-[3-dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDCI, 6.84g, 35.7 mmol), and DMAP (0.436 g, 3.57 mmol) in anhydrousdichloromethane (180 mL) was stirred at room temperature under an argonatmosphere for 2 days. The solution was diluted with dichloromethane andwashed with water (300 mL), 5% sodium bicarbonate solution (300 mL), andbrine (300 mL). After drying over sodium sulfate, filtration, andconcentration under reduced pressure, the remaining mixture (22.37 g)was purified by column chromatography on silica gel (600 g), elutingwith heptane and ethyl acetate (4:1). The product containing fractions(16.38 g) were combined and concentrated and purified a second time bycolumn chromatography on silica gel (1 Kg), eluting with heptane andethyl acetate (6:1). The product containing fractions were combined,concentrated, and dried under high vacuum. The protected L-serine esterof benazepril SPIB0050101 (15.4 g, 76% yield) was isolated as a whitefoam.

¹H NMR (300 MHz, CDCl₃): δ 7.30-7.10 (9H, m), 6.22 (1H, d, J=6.9 Hz),5.36 (1H, d, J=17.4 Hz), 4.98 (1H, m), 4.70 (1H, d, J=9.6 Hz), 4.58 (1H,m), 4.45 (1H, d, J=8.7 Hz), 4.28 (1H, m), 4.04 (1H, d, J=17.4 Hz), 3.32(1H, m), 2.90 (1H, m), 2.73-2.52 (3H, m), 2.06 (2H, m), 1.77 (1H, m),1.49 (9H, s), 1.41 (9H, s), 1.34 (18H, s).

¹³C NMR (75 MHz, CDCl₃): δ 171.59, 170.95, 168.83, 167.50, 155.86,154.14, 141.52, 140.07, 135.70, 129.35, 128.45, 128.31, 127.71, 126.89,125.86, 122.03, 82.03, 81.89, 81.02, 79.56, 64.89, 57.28, 53.64, 53.28,49.54, 36.19, 34.80, 34.31, 28.49, 28.32, 27.95.

HPLC analysis:

98.41% purity; r.t.=11.595 min.; 25% DIUF water/75% acetonitrile; 1mL/min; 40° C.; Synergi Polar-RP (serial #161309-2), 4.6×250 mm; 20 ulinjection.

Deprotection and Purification:

A solution of hydrogen chloride (1.0 M in acetic acid, 384 mL) was addeddrop-wise to a solution of the protected L-serine ester of benazeprilSPIB0050101 (15.3 g, 19.2 mmol) in anhydrous dichloromethane (240 mL)that was cooled in an ice batch, under an argon atmosphere. The mixturewas stored for 7 days at 5° C. The mixture was concentrated underreduced pressure and dried under high vacuum to generate a colorlessgel. Ethyl acetate (150 mL) was added to the gel and the mixture wassonicated for 5 minutes. The white, solid product was concentrated underreduced pressure. An additional amount of ethyl acetate (150 mL) wasadded to the solid and the mixture was heated to reflux for 10 minutes.The solution was cooled to room temperature. The solids were filteredand dried under high vacuum. The benazepril-serine ester salt (12.3 g)was purified by stirring the material in anhydrous dichloromethane (60mL) overnight, at room temperature. After filtration and drying, therecovered material (9.3 g) was purified a final time by stirring thematerial in ethyl acetate (60 mL) overnight, at room temperature. Thefinal salt was filtered and dried under high vacuum (at 45° C.)overnight. In order to remove traces of solvent the final salt (8.70 g)was dissolved in DIUF water (150 mL) and freeze-dried. Benazeprilatserine ester, dihydrochloride SPIB00501 (8.61 g, 80% yield, 99.69%purity by HPLC) was isolated in monohydrate form (as white solid).

¹H NMR (300 MHz, DMSO): δ 10.89 (4H, s, br), 8.88 (3H, s, br), 7.33-7.17(9H, m), 4.67 (1H, d, J=17.4 Hz), 4.47 (3H, m), 4.28 (1H, m), 3.83 (1H,m), 3.73 (1H, t, J=9.0 Hz), 3.23 (1H, m), 2.66 (3H, m), 2.52 (1H, m),2.26 (1H, m), 2.09 (2H, m).

¹³C NMR (75 MHz, DMSO): δ 169.76, 168.27, 168.11, 167.68, 140.51,139.72, 134.67, 129.29, 128.35, 128.24, 127.94, 126.88, 125.96, 123.14,62.85, 58.33, 56.34, 51.05, 50.29, 33.88, 31.78, 30.54, 26.87.

HPLC Analysis:

99.69% purity, r.t.=12.408 min, sample dissolved in DIUF water/ACN, 82%DIUF water (0.1% TFA)/18% ACN, Synergi Polar-RP (#258258-4), 4 u,250×4.6 mm, 1 mL/min., 30° C., 20 uL inj. vol., SPD-10Avp, chl-210 nm.

Specific rotation: −123.3 deg (20° C., 21.3 mg/2 mL ethanol, 589 nm).

Melting point: 148.0° C. decomposed (Thomas Hoover CMPA, uncorrected)

CHN Analysis:

calc.: C, 53.29; H, 5.85; N, 7.46, Cl, 10.70 (C₂₅H₂₉N₃O₇-1.7HCl—H₂O).found: C, 53.30, H, 5.86, N, 7.35, Cl, 10.78.

5) Preparation of the L-Threonine Ester of Benazepril CouplingProcedure:

A mixture of acid 4 (20.7 g, 37.5 mmol), boc-L-threonine tert-butylester (9.36 g, 34.0 mmol, prepared by the literature method)¹,1-[3-dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDCI, 9.12g, 47.6 mmol), and DMAP (0.582 g, 4.76 mmol) in anhydrousdichloromethane (250 mL) was stirred at room temperature under an argonatmosphere for 3 days. The reaction solution was washed with water(2×250 mL), 5% sodium bicarbonate solution (250 mL), and brine (250 mL).After drying over sodium sulfate, filtration, and concentration underreduced pressure, the remaining yellow oil (38.7 g) was purified bycolumn chromatography on silica gel (1 Kg), eluting with heptane-ethylacetate (4:1). The product containing fractions were combined andconcentrated. The L-threonine ester of benazepril SPIB0050201 (18.51 g,67% yield) was isolated.

¹H NMR (300 MHz, CDCl₃): δ 7.26-7.16 (9H, m), 5.63 (1H, s, br), 5.40(1H, s, br), 4.99 (2H, m), 4.11 (1H, d, J=15.6 Hz), 3.27 (1H, s, br),2.68 (2H, s, br), 2.56 (1H, dd, J=13.2, 6.0 Hz), 2.35 (2H, s, br), 2.05(2H, m), 1.47-1.26 (39H, m).

¹³C NMR (75 MHz, CDCl₃): δ 170.34, 169.72, 168.86, 167.71, 156.18,154.83, 141.23, 140.60, 135.58, 129.08, 128.41, 127.61, 126.53, 125.91,122.09, 82.26, 81.68, 80.85, 79.66, 71.64, 58.04, 55.76, 54.23, 50.01,35.20, 34.54, 33.87, 28.41, 28.28, 27.96, 17.28.

HPLC Analysis:

98.30% purity; r.t.=11.924 min.; 25% DIUF water/75% acetonitrile; 1mL/min; 40° C.; Synergi Polar-RP (serial #161309-2), 4.6×250 mm; 20 ulinjection, 210 nm.

Melting point: 74.8° C.

Deprotection and Purification:

A solution of hydrogen chloride (1.0 M in acetic acid, 452 mL) was addeddrop-wise to a solution of the protected L-threonine ester of benazeprilSPIB0050201 (18.3 g, 22.6 mmol) in anhydrous dichloromethane (250 mL)that was cooled in an ice batch, under an argon atmosphere. The reactionmixture was stored for 8 days at 5° C., then concentrated under reducedpressure and dried under high vacuum to generate white foam. Ethylacetate (200 mL) was added to the foam and the mixture was sonicated for5 minutes. The white, solid product was concentrated under reducedpressure. An additional amount of ethyl acetate (200 mL) was added tothe solid and the mixture was heated to reflux for 15 minutes. Aftercooling the solution to room temperature, the solids were filtered anddried under high vacuum (at 40° C.) until a constant weight wasachieved. The remaining salt (11.9 g) was purified by stirring inanhydrous dichloromethane (90 mL) for three days at room temperature.The salt was filtered and dried under high vacuum (at 45° C.) until aconstant weight was achieved. In order to remove traces of solvent thefinal salt (11.02 g) was suspended in DIUF water (200 mL) andfreeze-dried. The benazeprilat L-threonine ester, dihydrochlorideSPIB00502 (10.87 g, 81% yield, 97.55% purity by HPLC) was isolated indihydrate form (as a white solid).

¹H NMR (300 MHz, DMSO): δ 10.62 (3H, s, br), 8.85 (4H, s, br), 7.33-7.18(9H, m), 5.29 (1H, m), 4.65 (1H, d, J=17.1 Hz), 4.42 (1H, d, J=17.1 Hz),4.12 (1H, d, J=2.4 Hz), 3.84 (1H, m), 3.58 (1H, dd, J=8.7, 8.1 Hz), 3.23(1H, m), 2.67 (3H, m), 2.54 (1H, m), 2.26 (1H, m), 2.09 (2H, m), 0.97(3H, d, J=6.3 Hz).

¹³C NMR (75 MHz, DMSO): δ 169.73, 167.81, 167.35, 166.45, 140.28,139.67, 134.59, 129.33, 128.36, 127.97, 127.04, 126.11, 123.14, 69.75,58.70, 56.21, 55.40, 50.07, 33.58, 31.97, 30.64, 26.73, 16.09.

HPLC Analysis:

97.55% purity, r.t.=15.517 min, sample dissolved in DIUF water/ACN, 82%DIUF water (0.1% TFA)/18% ACN, Synergi Polar-RP (#258258-4), 4 u,250×4.6 mm, 1 mL/min., 35° C., 20 uL inj. vol., SPD-10Avp, chl-210 nm.

Specific rotation: −134 deg (21° C., 21.6 mg/2 mL ethanol, 589 nm)

Melting point: 152.0-153.0° C. (Thomas Hoover CMPA, uncorrected)

CHN Analysis:

calc.: C, 52.59; H, 6.15; N, 7.08, Cl, 10.75 (C₂₆H₃₁N₃O₇-1.8HCl-1.7H₂O).found: C, 52.81, H, 6.15, N, 7.37, Cl, 10.79.

6) Preparation of the L-Hydroxyproline Ester of Benazepril CouplingProcedure:

A mixture of acid 4 (35.8 g, 64.8 mmol), boc-L-hydroxyproline tert-butylester (16.9 g, 58.9 mmol, prepared by the literature method),1-[3-dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDCI, 15.8g, 82.5 mmol), and DMAP (1.01 g, 8.25 mmol) in anhydrous dichloromethane(600 mL) was stirred at room temperature under an argon atmosphere for 6days. The dichloromethane solution was washed with water (400 mL), 5%sodium bicarbonate solution (400 mL), and brine (400 mL). After dryingover sodium sulfate, filtration, and concentration under reducedpressure, the remaining brown oil (51.5 g) was purified by columnchromatography on silica gel (1 Kg), eluting with heptane-ethyl acetate(4:1). The product containing fractions were combined and concentrated.The protected L-hydroxyproline ester of benazepril SPIB0050301 (19.69 g,41% yield) was isolated as white foam.

¹H NMR (300 MHz, CDCl₃): δ 7.27-7.12 (9H, m), 5.33 (1H, m), 4.85 (2H,dd, J=17.1, 5.4 Hz), 4.36 (1H, m), 4.23 (1H, t, J=7.50 Hz), 4.10 (1H,dd, J=17.1, 3.6 Hz), 3.73 (1H, m), 3.65 (1H, m), 3.24 (1H, m), 2.71 (2H,m), 2.57-2.40 (3H, m), 2.21-1.97 (4H, m), 1.44 (36H, m), 1.47-1.37 (36H,m).

¹³C NMR (75 MHz, CDCl₃): δ 171.53, 171.42, 170.90, 170.84, 170.59,167.62, 154.61, 153.90, 153.48, 141.29, 141.25, 140.69, 135.46, 129.13,128.43, 128.34, 127.71, 126.58, 125.92, 122.20, 81.72, 81.24, 80.78,80.03, 79.88, 73.04, 72.25, 58.52, 58.04, 54.58, 52.03, 51.67, 50.41,36.72, 35.58, 34.52, 33.93, 28.40, 28.34, 28.04, 27.99.

Deprotection and Purification:

A solution of hydrogen chloride (1.0 M in acetic acid, 400 mL) was addeddrop-wise to a solution of the protected L-hydroxyproline ester ofbenazepril SPIB0050301 (19.69 g, 24.0 mmol) in anhydrous dichloromethane(265 mL) that was cooled in an ice batch, under an argon atmosphere. Thereaction mixture was stored for 4 days at 5° C. and then wasconcentrated under reduced pressure and dried under high vacuum togenerate a light, yellow foam. Ethyl acetate (200 mL) was added to theyellow foam and the mixture was sonicated for 5 minutes. The white,solid product was concentrated under reduced pressure. An additionalamount of ethyl acetate (200 mL) was added to the solid and the mixturewas heated to reflux for 15 minutes. The solution was cooled to roomtemperature. The solids were filtered and dried under high vacuum at 35°C. The remaining solid (14.50 g) was purified by stirring in anhydrousdichloromethane (60 mL) overnight at room temperature. The remainingsalt (13.19 g) was filtered and dried under high vacuum at 45° C. for 20hours. In order to remove trace of solvent, the salt was dispersed inDIUF water (500 mL) and freeze-dried. Benazeprilattrans-4-hydroxy-L-proline ester, dihydrochloride SPIB00503 (12.5 g, 87%yield, 99.95% purity by HPLC) was isolated in monohydrate form (as awhite solid).

¹H NMR (300 MHz, DMSO): δ 10.34 (6H, s, br), 7.34-7.17 (9H, m), 5.25(1H, m), 4.66 (1H, d, J=17.4 Hz), 4.43 (1H, d, J=17.4 Hz), 4.24 (1H, t,J=9.0 Hz), 3.88 (1H, m), 3.77 (1H, t, J=9.0 Hz), 3.51 (1H, dd, J=12.6,3.9 Hz), 3.23 (2H, m), 2.66 (4H, m), 2.22 (3H, m), 2.11 (2H, m).

¹³C NMR (75 MHz, DMSO): δ 169.80, 169.01, 167.93, 167.88, 140.40,139.76, 134.59, 129.17, 128.33, 128.27, 127.87, 126.87, 126.01, 123.13,74.10, 57.88, 57.39, 56.31, 50.23, 49.95, 33.98, 33.69, 31.68, 30.60,26.79.

HPLC Analysis:

99.95% purity, r.t.=16.725 min, sample dissolved in DIUF/ACN, 82% DIUFwater (0.1% TFA)/18% ACN, Synergi Polar-RP (#258258-4), 4 u, 250×4.6 mm,1 mL/min., 30° C., 20 uL inj. vol., SPD-10Avp, chl-210 nm.

Specific rotation: −116.0 deg (20° C., 23.7 mg/2 mL ethanol, 589 nm)

Melting point: 168.0° C. decomposed (Thomas Hoover CMPA, uncorrected)

CHN Analysis:

calc.: C, 55.52; H, 5.96; N, 7.19, Cl, 9.41 (C₂₇H₃₁N₃O₇-1.55HCl—H₂O).found: C, 55.55, H, 5.99, N, 7.16, Cl, 9.67.

Literature Cited Moore, J. W.; Szelke, M. Tetrahedron Lett. 1970,4423-4426.

Synthesis and Efficacy Studies of Enalapril Drivatives: Overview:

The procedure for the synthesis of the L-serine, L-threonine, andL-hydroxyproline esters of enalapril is outlined in Synthetic Sequencesection. The complete procedure and analytical data are given in theExperimental Section. In general, the maleate salt of enalapril(Xiangding Chemical International Company) was butylated to protect thefree acid group. The ethyl ester was then hydrolyzed to the acid withsodium hydroxide, followed by acidification. The amine present in themolecule was protected with a tert-butyloxycarbonyl group to preventdimerization during the subsequent coupling reaction. The protectedintermediate was then coupled with the tert-butyl ester of boc-L-serine,boc-L-threonine, or boc-L-hydroxyproline using EDC. The protectedintermediate esters were treated with dilute hydrochloric acid in aceticacid at low temperature to remove all of the protecting groups. Washingwith ethyl acetate and dichloromethane (or ACN) purified the amino acidester salts of Enalapril. The final salts were dried under high vacuumand analyzed for NMR, HPLC, CHN, specific rotation, and melting point.

Synthetic Sequence:

Synthesis of the L-Serine, L-Threonine, and L-Hydroxyproline Esters ofEnalapril a) THF; b) NaOH, MeOH, H₂O; c) (Boc)₂O, Et₃N, CH₂Cl₂; d)Boc-Ser-OtBu, EDC, DMAP, CH₂Cl₂; e) Boc-Thr-OtBu, EDC, DMAP, CH₂Cl₂; f)Boc-Hyp-OtBu, EDC, DMAP, CH₂Cl₂; g) HCl, AcOH, CH₂Cl₂ ExperimentalSection

The synthesis of SPIB00401, SPIB00402 and SPIB00403 was conducted inbatches. Reagents mentioned in the experimental section were purchasedat the highest obtainable purity from Lancaster, Sigma-Aldrich, Acros,Bachem, or Xiangding Chemical International, except for solvents, whichwere purchased from either Fisher Scientific or Mallinkrodt.

1) Butylation of Enalapril:

A solution of 2-tert-butyl-1,3-dicyclohexyl isourea 1 (592 g, 2.11 mol,prepared by the literature procedure) in THF (500 mL) was addeddrop-wise to a mixture of enalapril maleate (130 g, 0.264 mol) inanhydrous THF (600 mL) cooled in an ice-water bath. The mixture wasallowed to stir for 24 hours at room temperature under an argonatmosphere. The reaction mixture was filtered, and the precipitate (DCU)was washed with dichloromethane (3×500 mL). The filtrates were combinedand concentrated to produce a green oil. The residue was diluted withdichloromethane (1 L), washed with 5% sodium bicarbonate solution (1 L),and washed with brine (500 mL). The dichloromethane solution was driedover sodium sulfate, filtered, and concentrated under reduced pressure.The remaining mixture (189 g) was purified by column chromatography onsilica gel (1 Kg), eluting with heptane-ethyl acetate (2:1). The productcontaining fractions were combined and concentrated under reducedpressure and dried at high vacuum. Butylated enalapril 2 (87.3 g, 76%yield) was obtained as a light yellow oil.

¹H NMR (300 MHz, CDCl₃): δ 7.31-7.15 (5H, m), 4.43 (1H, dd, J=8.4, 3.3Hz), 4.18 (2H, q, J=7.2 Hz), 3.52 (3H, m), 3.25 (1H, t, J=6.6 Hz), 2.67(2H, m), 2.24 (1H, s, br), 2.18 (1H, m), 1.98 (5H, m), 1.45 (9H, s),1.29 (3H, d, J=7.2 Hz), 1.25 (3H, t, J=7.2 Hz).

¹³C NMR (75 MHz, CDCl₃): δ 173.99, 173.03, 170.81, 141.07, 128.15,128.09, 125.68, 81.03, 60.59, 59.93, 59.44, 53.36, 46.41, 35.06, 31.95,28.86, 27.88, 24.72, 18.80, 14.28.

2) Saponification of the Butylated Enalapril:

A sodium hydroxide solution (80.8 g, 2.02 mol) in DIUF water (1 L) wasadded in a drop-wise fashion to the butylated enalapril 2 (87.2 g, 0.202mol) dissolved in methanol (1 L), while cooling the methanol solution inan ice bath. The solution was allowed to stir for 18 hours under anargon atmosphere while cooling in an ice bath. The reaction mixture wasconcentrated under reduced pressure and dried under high vacuum toproduce a cloudy solution (847 g). The residue was cooled in an ice bathand a solution of acetic acid in water (1.2 L, 1:10 v/v aceticacid/water) was added drop-wise to adjust the solution to pH=7. A whiteprecipitate formed which was extracted with dichloromethane (4×500 mL).The combined dichloromethane fractions were dried over sodium sulfate,filtered, and concentrated under reduced pressure to generate a brownfoam-like solid (73.4 g). After additional drying under high vacuum, 3(61.5 g, 75% yield) was obtained as a light brown solid, which was usedwithout additional purification.

¹H NMR (300 MHz, CDCl₃): δ 7.12 (5H, m), 5.31 (2H, s, br), 4.41 (1H, dd,J=9.0, 3.6 Hz), 3.97 (1H, q, J=6.6 Hz), 3.53 (1H, m), 3.42 (1H, m), 3.31(1H, t, J=6.3 Hz), 2.72 (2H, m), 2.21-1.91 (6H, m), 1.52 (3H; d, J=6.6Hz), 1.43 (9H, s).

¹³C NMR (75 MHz, CDCl₃): δ 172.66, 170.29, 168.74, 140.46, 128.30,128.18, 125.80, 81.63, 61.74, 59.75, 54.57, 46.64, 35.53, 31.89, 28.90,27.97, 24.61, 16.47.

3) Amine Protection:

A solution of di-tert-butyl dicarbonate (36.5 g, 167 mmol) in anhydrousdichloromethane (200 mL) was added in a drop-wise fashion to a solutionof 3 (61.5 g, 152 mmol) and triethylamine (61.5 g, 608 mmol) inanhydrous dichloromethane (600 mL) cooled in an ice bath, under an argonatmosphere. The mixture was allowed to stir at room temperature for 3days at room temperature under an argon atmosphere. The reaction mixturewas washed with ice-cold 0.5N hydrochloric acid (1.2 L) and brine (600mL). The dichloromethane solution was dried over sodium sulfate,filtered, and concentrated under reduced pressure. The remaining yellowoil (87 g) was purified by column chromatography on silica gel (1 Kg),eluting with heptane-ethyl acetate (3:1). The product containingfractions were combined, concentrated under reduced pressure, and driedunder high vacuum until the weight was constant. A protected amine 4(28.7 g, 37% yield) was obtained as a white solid.

¹H NMR (300 MHz, CDCl₃): δ 13.61 (1H, br s), 7.30-7.16 (5H, m), 4.79(1H, q, J=7.5 Hz), 4.47 (1H, dd, J=8.7, 4.5 Hz), 3.75 (2H, m), 3.54 (1H,m), 2.84 (2H, m), 2.68 (1H, m), 2.22 (1H, m), 1.98 (4H, m), 1.43 (9H,s), 1.10 (3H, d, J=7.5 Hz).

¹³C NMR (75 MHz, CDCl₃): δ 174.62, 172.12, 170.10, 154.70, 141.23,128.57, 128.41, 126.00, 82.74, 81.81, 60.00, 55.63, 53.00, 46.89, 34.27,33.40, 28.80, 28.16, 27.99, 24.86, 14.08.

4) Preparation of the L-Serine Ester of Enalapril: Coupling Procedure:

A mixture of 4 (15.3 g, 30.3 mmol), boc-L-serine tert-butyl ester (7.19g, 27.5 mmol, prepared by the literature method),1-[3-dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDCI, 7.38g, 38.5 mmol), and DMAP (0.47 g, 3.85 mmol) in anhydrous dichloromethane(200 mL) was stirred at room temperature under an argon atmosphere for 4days. The dichloromethane solution was washed with water (2×200 mL) andbrine (200 mL). After drying the dichloromethane solution over sodiumsulfate, filtration, and concentration under reduced pressure, theresidue (24.6 g) was purified by column chromatography on silica gel(700 g), eluting with heptane-ethyl acetate (4:1). The productcontaining fractions were combined and concentrated. The remaining whitesolid (14.1 g) was crystallized from heptane (100 mL) and methyltert-butyl ether (50 mL). After filtration and drying, the protectedL-serine ester of enalapril SPIB0040101 (13.5 g, 66% yield) was obtainedas a white solid.

¹H NMR (300 MHz, CDCl₃): δ 7.29 (2H, m), 7.20 (3H, m), 5.55 (1H, d, Hz),5.26 (1H, m), 4.50-4.23 (5H, m), 3.79 (1H, m), 3.66 (1H, m), 2.76 (1H,m), 2.58 (2H, m), 2.25 (1H, m), 1.97 (4H, m), 1.44 (36H, m), 1.27 (3H,d, J=6.6 Hz).

¹³C NMR (75 MHz, CDCl₃): δ 171.11, 169.36, 168.33, 155.18, 154.32,141.43, 128.21, 128.19, 125.78, 82.48, 80.98, 80.86, 79.75, 65.05,59.58, 55.70, 53.34, 50.12, 47.00, 35.20, 34.19, 29.14, 28.35, 28.27,27.96, 27.90, 24.50, 16.55.

HPLC Analysis:

98.81% purity; r.t.=9.433 min.; 25% DIUF water/75% acetonitrile; 1mL/min; 39° C.; Synergi Polar-RP (serial #234257-1), 4.6×250 mm; 20 ulinjection; RI 210 nm.

Melting point: 134-135° C. (Thomas Hoover CMPA, uncorrected)

Deprotection and Purification:

A solution of hydrogen chloride (1.0 M in acetic acid, 360 mL) was addeddrop-wise to a solution of the protected L-serine ester of enalaprilSPIB0040101 (13.4 g, 17.9 mmol) in anhydrous dichloromethane (190 mL)that was cooled in an ice batch, under an argon atmosphere. The mixturewas stored for 8 days at 5° C. The mixture was concentrated underreduced pressure and dried under high vacuum to generate a light, yellowoil (19.56 g). Ethyl acetate (100 mL) was added to the oil and themixture was sonicated for 5 minutes. The white, solid product (14.8 g)was filtered and dried under high vacuum. An additional amount of ethylacetate (200 mL) was added to the solid and the mixture was heated toreflux for 10 minutes. While the solution was cooling, the solid wassonicated for 30 minutes and stirred for 2 hours at room temperature.The solids were filtered and dried under high vacuum. The remaining salt(10.5 g) was purified twice by stirring the material in anhydrousdichloromethane (60 mL) overnight, at room temperature. The final saltwas filtered and dried under high vacuum at room temperature until aconstant weight was obtained. Enalaprilic acid L-serine ester,dihydrochloride SPIB00401 (8.01 g, 88% yield, 97.09% purity by HPLC) wasobtained in monohydrate form (as white solid).

¹H NMR (300 MHz, DMSO): δ 10.76 (4H, s, br), 9.02 (3H, s, br), 7.25 (5H,m), 4.67 (2H, m), 4.35 (3H, m), 3.93 (1H, m), 3.70 (1H, m), 3.42 (1H,m), 2.62 (2H, m), 2.17 (3H, m), 1.89 (3H, m), 1.44 (3H, d, J=6.0 Hz).

¹³C NMR (75 MHz, DMSO): δ 172.53, 167.89, 167.03, 166.94, 140.26,128.33, 126.11, 63.21, 58.90, 58.44, 53.97, 51.13, 46.83, 31.44, 30.45,28.65, 24.71, 15.35.

HPLC Analysis:

97.09% purity; r.t.=8.367 min.; 85% DIUF water (0.1% TFA)/15% ACN; 1mL/min; 36.0 C; Synergi Polar-RP 5 u column (serial #161309-2), 4.6×250mm; 20 ul injection; Sig=210.4.

HRMS [M-2HCl—H₂O+H]: calc. 436.2084 (C₂₁H₃₁Cl₂N₃O₇). found 436.2079.

Melting point: 129.5° C. decomposed (Thomas Hoover CMPA, uncorrected)

CHN Analysis:

calc.: C, 47.91; H, 6.32; N, 7.98 (C₂₁H₃₁Cl₂N₃O₇—H₂O). found: C, 47.95;H, 6.32; N, 7.83.

5) Preparation of the L-Threonine Ester of Enalapril: CouplingProcedure:

A mixture of 4 (28.4 g, 56.3 mmol), boc-L-threonine tert-butyl ester(14.1 g, 51.2 mmol, prepared by the literature method),1-[3-dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDCI, 13.7g, 71.7 mmol), and DMAP (0.876 g, 7.17 mmol) in anhydrousdichloromethane (450 mL) was stirred at room temperature under an argonatmosphere for 13 days. The dichloromethane solution was washed withwater (2×500 mL) and brine (500 mL). After drying over sodium sulfate,filtration, and concentration under reduced pressure, the remainingyellow oil (46.49 g) was purified by column chromatography on silica gel(1 Kg), eluting with heptane-ethyl acetate (5:1). The product containingfractions were combined in two fractions and concentrated. ProtectedL-threonine ester of enalapril SPIB0040201 (7.35 g and 6.83 g, 36.3%yield) was present in both fractions. Both fractions were deprotected asdescribed below and recombined in the final purification.

¹H NMR (300 MHz, CDCl₃): δ 7.30 (2H, m), 7.21 (3H, m), 5.28 (2H, m),5.14 (1H, d, J=8.4 Hz), 4.30-4.22 (3H, m), 3.85 (1H, m), 3.65 (1H, m),2.65 (2H, m), 2.39 (1H, m), 2.20 (1H, m), 1.99 (4H, m), 1.46 (36H, m),1.25 (6H, m).

¹³C NMR (75 MHz, CDCl₃): δ 171.23, 170.89, 170.03, 168.57, 155.82,154.64, 141.43, 128.37, 128.24, 125.92, 82.83, 81.06, 80.01, 79.73,72.23, 68.34, 59.80, 59.20, 57.69, 55.65, 50.78, 47.08, 34.40, 29.14,28.41, 28.30, 28.03, 24.67, 20.01, 17.15.

Deprotection and Purification:

A solution of hydrogen chloride (1.0 M in acetic acid, 185 mL) was addeddrop-wise to a solution of one of the batches of the protectedL-threonine ester of enalapril SPIB0040201 (7.05 g, 9.25 mmol batch 1,6.63 g; 8.70 mmol, batch 2) in anhydrous dichloromethane (100 mL) thatwas cooled in an ice batch, under an argon atmosphere. The mixture wasstored for 7 days at 5° C. The mixture was concentrated under reducedpressure and dried under high vacuum to generate a colorless gel. Ethylacetate (250 mL) was added to the gel and the mixture was sonicated for5 minutes. The white, solid product was filtered and dried under highvacuum. An additional amount of ethyl acetate (100 mL) was added to thesolid and the mixture was heated to reflux for 10 minutes. After coolingthe solution to room temperature, the solids were filtered and driedunder high vacuum until a constant weight was achieved. The deprotectionstep was then repeated with the second batch of material.

Both batches of the final salt (4.84 g from batch 1 and 4.30 from batch2) were combined and purified by stirring in anhydrous dichloromethane(60 mL) overnight at room temperature. The final salt was filtered anddried under high vacuum. The washing procedure was then repeated for twodays with ethyl acetate (40 mL) and for 18 hours with acetonitrile (50mL). After filtration and drying, enalaprilic acid L-threonine ester,dihydrochloride SPIB00402 (7.75 g, 82% yield, 97.61% purity by HPLC) wasisolated in monohydrate form (as white solid).

¹H NMR (300 MHz, DMSO): δ 10.50 (3H, s, br), 8.96 (4H, s, br), 7.24 (5H,m), 5.44 (1H, m), 4.58 (0.3H, m), 4.34-3.90 (3.4H, m), 3.78 (0.3H, m),3.64 (0.7H, m), 3.47 (1H, m), 3.29 (0.3H, m), 2.72 (1H, m), 2.52 (1H,m), 2.16 (3H, m), 1.87 (2.7H, m), 1.60 (0.3H, m), 1.44 (6H, m).

¹³C NMR (75 MHz, DMSO): δ 173.21, 172.55, 168.02, 167.88, 166.99,166.71, 140.36, 128.37, 126.14, 70.43, 70.22, 58.96, 58.25, 58.13,55.56, 54.85, 53.63, 46.65, 46.48, 32.13, 31.55, 30.47, 28.67, 24.70,21.88, 16.99, 16.56, 16.50, 15.12.

HPLC Analysis:

97.61% purity, r.t.=9.258 min, sample dissolved in DIUF water/ACN, 85%DIUF water (0.1% TFA)/15% ACN, Synergi Polar-RP (#161309-2), 4 u,250×4.6 mm, 1 mL/min., 40° C., 20 uL inj. vol., SPD-10Avp, chl-210 nm.

Specific rotation: −33.8 deg (21° C., 17.3 mg/2 mL ethanol, 589 nm)

Melting point: 147.0-148.0° C. decomposed (Thomas Hoover CMPA,uncorrected)

CHN Analysis:

calc.: C, 49.90; H, 6.61; N, 7.94, Cl, 1.38 (C₂₂H₃₁N₃O₇-1.7HCl—H₂O).found: C, 50.12, H, 6.61, N, 7.90, Cl, 1.33.

6) Preparation of the L-Hydroxyproline Ester of Enalapril: CouplingProcedure:

A mixture of 4 (24.6 g, 48.7 mmol), boc-L-hydroxyproline tert-butylester (12.7 g, 44.3 mmol, prepared by the literature method),1-[3-dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (EDCI, 11.9g, 62.0 mmol), and DMAP (0.757 g, 6.20 mmol) in anhydrousdichloromethane (400 mL) was stirred at room temperature under an argonatmosphere for 9 days. The dichloromethane solution was washed withwater (400 mL), 5% sodium bicarbonate solution (400 mL), water (400 mL),and brine (400 mL). After drying the dichloromethane solution oversodium sulfate, filtration, and concentration under reduced pressure,the residual green oil (42.0 g) was purified by column chromatography onsilica gel (1 Kg), eluting with heptane-ethyl acetate (5:1). The productcontaining fractions were combined, concentrated, and dried under highvacuum till a constant weight was obtained, yielding protectedL-hydroxyproline ester of enalapril SPIB0040301 (11.41 g, 33% yield).

¹H NMR (300 MHz, CDCl₃): δ 7.29 (2H, m), 7.19 (3H, m), 5.24 (2H, m),4.21 (3H, m), 3.74 (3H, m), 3.50 (1H, m), 2.77 (1H, m), 2.67-2.50 (2H,m), 2.23 (3H, m), 1.98 (4H, m), 1.44 (36H, m), 1.30 (3H, m).

¹³C NMR (75 MHz, CDCl₃): δ 171.09, 170.91, 169.08, 154.37, 153.80,153.44, 141.49, 128.37, 128.27, 125.91, 81.48, 81.30, 81.10, 80.32,73.15, 72.46, 59.78, 58.49, 55.48, 51.95, 51.65, 50.36, 47.27, 36.74,35.60, 35.17, 34.32, 29.29, 28.40, 28.31, 28.03, 24.70, 16.45.

Deprotection and Purification:

A solution of hydrogen chloride (1.0 M in acetic acid, 290 mL) was addeddrop-wise to a solution of the protected L-hydroxyproline ester ofenalapril SPIB0040301 (11.23 g, 14.5 mmol) in anhydrous dichloromethane(160 mL) that was cooled in an ice batch, under an argon atmosphere. Themixture was stored for 8 days at 5° C. The mixture was concentratedunder reduced pressure and dried under high vacuum to generate yellowfoam. Ethyl acetate (100 mL) was added to the yellow foam, and themixture was sonicated for 5 minutes. The white, solid product wasfiltered and dried under high vacuum. An additional amount of ethylacetate (100 mL) was added to the solid, and the mixture was heated toreflux for 10 minutes. After cooling the solution to room temperature,the solids were filtered and dried under high vacuum until a constantweight was achieved. The remaining solid (6.89 g) was purified bystirring in anhydrous dichloromethane (60 mL) overnight at roomtemperature. The remaining salt (5.80 g) was filtered and dried underhigh vacuum. In order to remove trace of solvent, the salt was dissolvedin water (100 mL) and freeze-dried. Enalaprilic acidtrans-4-hydroxy-L-proline ester, dihydrochloride SPIB00403 (5.40 g, 67%yield, 99.68% purity by HPLC) was obtained in monohydrate form (as whitesolid).

¹H NMR (300 MHz, DMSO): δ 10.65 (6H, s, br), 7.32-7.19 (5H, m), 5.44(0.3H, m), 5.36 (0.7H, m), 4.67 (0.3H, m), 4.44 (1H, t, J=8.7 Hz), 4.28(1.7H, m), 3.96 (1H, m), 3.73-3.31 (4H, m), 2.78 (1H, m), 2.61 (1H, m),2.33 (1H, m), 2.18 (3.3H, m), 1.93-1.59 (3.7H, m), 1.46 (3H, m).

¹³C NMR (75 MHz, DMSO): δ 173.31, 172.50, 169.24, 167.85, 167.66,167.21, 140.30, 140.22, 128.27, 128.22, 126.01, 74.64, 58.89, 58.70,57.92, 57.74, 57.58, 57.39, 54.68, 53.96, 50.31, 50.09, 46.59, 46.41,34.16, 34.06, 31.93, 31.34, 30.34, 28.55, 24.62, 21.84, 16.53, 15.20.

HPLC Analysis:

99.68% purity, r.t.=6.700 min, sample dissolved in DIUF water/ACN, 82%DIUF water (0.1% TFA)/18% ACN, Synergi Polar-RP (#258258-4), 4 u,250×4.6 mm, 1 mL/min., 35° C., 20 uL inj. vol., SPD-10Avp, chl-210 nm.

Specific rotation: −36.2 deg (21° C., 16.8 mg/2 mL ethanol, 589 nm)

Melting point: 159.0-160.0° C. (Thomas Hoover CMPA, uncorrected)

CHN analysis:

calc.: C, 50.01; H, 6.47; N, 7.61, Cl, 1.87 (C₂₃H₃₁N₃O₇-1.85HCl-1.3H₂O).found: C, 50.11, H, 6.57, N, 7.47, Cl, 1.84.

Effect of Various ACE Inhibitor Enalapril Derivatives on Hemodynamics inSpontaneously Hypertensive rats (SHRs).

ACE inhibitors have been known to be useful in antihypertensive therapyfor many years. Some ACE inhibitors are delivered orally as the parentdrug (captopril) while others are delivered as pro-drugs in order toobtain clinically useful plasma concentrations. This study provides dataon the effect of these various enalapril derivatives on hemodynamics inSHRs when given orally and compared to enalapril.

Methods

Briefly, SHRs fitted with right carotid catheters were orally gavagedwith 100 mg/kg enalapril or equivalent does of the enalaprilderivatives, and the blood pressures were followed continuously up to 3hours and then a measurement at 24 hours. The animals were consciousthroughout the procedure and were placed in a restrainer and hooked upto a pressure transducer and a signal processor. After 3 hours, theanimals were placed back into their cage, and blood pressure was finallymeasured again at 24 hours.

Results

Enalapril reduced blood pressure at 3 hours and 24 hours byapproximately 20% depending on if data from systolic, diastolic or meanblood pressure were analyzed. The pressures generally stayed at baselinelevels up to the 3 hour time point, and the pressure at 3 hours wasremarkably similar to that seen at 24 hours. Vehicle treated animalsshowed no change in blood pressure throughout the study.

The reduction in blood pressures caused by the various agents wasassociated with a slightly greater drop in diastolic pressure than insystolic pressure. For the purpose of comparison, mean arterial bloodpressures are utilized for this project summary. For the enalaprilseries, enalalpril and the amino acid derivatives of enalapril reducedarterial blood pressure.

No reflex tachycardia was noted and this is not unusual for ACEinhibitors. Heart rates tended to come down with time in all groups asthe animals became more accustomed to the restrainers.

VII Water Soluble Derivatives of Fibric Acid Derivatives

Fibric acid compounds are useful anti-hyperlipidemic drugs useful in thetreatment of hyperlipidemia in mammals where the symptoms are elevatedtriglycerides, low HDL (High density lipoproteins or “good” cholesterol,and elevated cholesterol. Fibric Acid compounds are also useful inreducing LDL (Low density lipoproteins, or “bad” cholesterol). Thegeneral structure of the fibric acid analogs is represented below, whereX is various mixed aliphatic and aromatic functionalities. Specificcompounds included in this formula are clofibric acid, fenofibric acid,ciprfibrate and gemfibrozil and the like.

Typical examples of the chemical moiety X in the above structure areshown below.

As used herein, the term “fibric acid compounds” refers to the fabricacid analogs depicted here which are not bonded to an amino acid. Whenthe fibric acid compounds are bonded to an amino acid, they will bereferred to as fibric acid “amino acid derivatives” or like term.

Fibric acid analogs shown in the structure above have been shown to havea large number of therapeutic applications, which are quite diverse andsomewhat surprising. Broadly, these compounds are useful in thetreatment dyslipidemia and dyslipoproteinemia. Dyslipidemia anddyslipoproteinemia are herein defined to include the group selected fromhypercholesterolemia, abnormal and elevated levels of cholesterol,abnormal and elevated levels of LDL cholesterol, abnormal and elevatedlevels of total cholesterol, abnormal and elevated levels of plasmacholesterol, abnormal and elevated levels of triglycerides,hypertrigylceridaemia, abnormal levels of lipoproteins, abnormal andelevated levels of low density lipoproteins (LDLs), abnormal andelevated levels of very low density lipoproteins, abnormal and elevatedlevels of very low intermediate density lipoproteins, abnormal levels ofhigh density lipoproteins, hyperlipidemia, hyperchylomicronemia,abnormal levels of chylomicrons, related disorders, and combinationsthereof such as those described in The ILIB Lipid Handbook for ClinicalPractice, Blood Lipids and Coronary Heart Disease, Second Edition, A. M.Gotto et al, International Lipid Information Bureau, New York, N.Y.,2000, the contents of which is hereby incorporated by reference.

Mechanism of Action:

The mechanism of action of Fibric acid compounds seen in clinicalpractice have been explained in-vivo in transgenic mice and in vitro inhuman hepatocytes cultures by the activation of peroxisome proliferatoractivated receptor alpha (PPAR-alpha). Through this mechanism, Fibricacid compounds increase lipolysis and elimination of triglyceride-richparticles from plasma by activating lipoprotein lipase and reducingproduction of apoprotein C-III (an inhibitor of lipoprotein lipaseactivity).

The resulting fall in triglycerides produces an alteration in the sizeand composition of LDL from small, dense particles (which are thought tobe atherogenic due their susceptibility to oxidation), to large buoyantparticles. These larger particles have greater affinity for cholesterolreceptors and are catabolized rapidly. Activation of PPAR-alpha alsoinduces an increase in the synthesis of apoproteins A-I, A-II, and HDLcholesterol. The Fibric Acid compounds depicted hereinabove are alsouseful in the treatment of gout, as they reduce serum uric acid levelsin hyperurecemic patients.

Hyperlipidemia types include type I, type IIa, type IIb, type III, typeIV, and type V. These types can be characterized according to the levelsrelative to normal levels of lipids (cholesterol and triglycerides) andlipoproteins in patients. Different classifications are derived fromDrug Facts and Comparisons, 52nd Edition (1998) page 1066 which ishereby incorporated by reference.

Many of the fibric acid compounds when administered orally do not havesufficient bioavilability, and furthermore absorption is variable anderratic and is dependent upon the intake of food. In fact absolutebioavialability of many of the fibric acid compounds is not possiblesince the ones currently marketed are insoluble in water, hence aparenteral formuation is difficult to prepare or not available.Furthermore, since these drugs usually are administered as esters, theyhave to be metabolized in the body to release active drug, which are thefibric acids. However, due to the ester formation of these drugs, theyare quite insoluble in water, hence are difficult to formulate, and arenot easily broken down in the body to release active drugs.

Many of the Fibric acid compounds are low to medium molecular weightsolids with characteristic odor. Taken orally they have unpleasant tasteand can severely irritate mouth and throat. Taken with food providesmore blood concentration compared to fasting. Overall bioavailabilityhas been reported anywhere between 40-60 and quite variable amongpatients. As shown hereinbelow, this fed fast difference inbioavailability is more pronounced when the Fibric acid compounds arecompared with that of the amino acid derivatives of Fibric acids.

One of the significant problems associated with currently marketedfibric acid compounds is that they are administered as prodrugs topatients which are metabolized to cleave off the prodrug moiety, e.g.,esters or alcohol and the cleaved products may themselves be highlytoxic. For example, in the case of fenofibrate and gemfibrozil,isopropyl alcohol is released as the esterase enzyme cleave thepro-moiety from the fenofibric acid. It is well known that isopropanolis highly toxic when released into any of the mammalian tissues.

In order to improve the therapeutic effectiveness, uniform bloodprofile, develop pharmaceutically elegant formulation and improve thesolubility of the drug in water, the present invention providesalternative derivatives of Fibric acid compounds which overcome many ofthe difficulties stated above.

Accordingly, in one aspect, the present invention is directed toalternate class of derivatives of Fibric acid compounds, namely aminoacid derivatives. The amino acid derivative consists of the hydroxylgroup of an amino acid esterified to the free carboxyl group present onthe Fibric acid compounds. In another embodiment, the amine group of theamino acid is reacted with COOH of the fibric acids to form an amidelinkage.

More specifically, in one aspect of the present invention is directedto, the compounds of the formulas

where x is as defined hereinaboveor pharmaceutically acceptable salts thereof; wherein R is either NH-AAor O-AA and AA is an amino acid, without the amino group or hydroxygroup, in which either an amine group or the hydroxyl group,respectively, is reacted with the carboxylic acid group of Fibric acidcompounds.

The present invention is also directed to a pharmaceutical compositioncomprising a therapeutically effective amount of the various Fibric acidamino acid derivatives and a pharmaceutical carrier therefor.

In another embodiment, the present invention is directed to a method oftreating a patient in need of Fibric acid therapy, which methodcomprises administering to said patient an effective amount of theFibric acid amino acid derivatives.

In a further embodiment, the present invention is directed to a methodof converting liquid Fibric acid compounds into a solid powder byreacting the carboxyl functionality of the Fibric acid compounds witheither amine or hydroxyl functionality of an amino acid undo conditionssufficient to form a covalent bond between the amino acid and the fibricacid compound and isolating the product thereof.

In a still further embodiment, the present invention is directed to amethod of substantially and in a therapeutically efficacious manner,administer the fibric acid amino acid derivatives which in this formfacilites the absorption of the fibric acid moiety, thereby improvingthe consistent therapeutic effect which method comprises administeringto a patient the amino acid derivative of fibric acid, prepared by aprocess which comprises reacting the COOH functionality of the Fibricacid compound with an amino acid, especially either NH₂ of the aminoacid or OH functionality of the hydroxy containing amino acids to forman amide or ester covalent bond respectively and isolating the productthereof.

The present inventor has found that amino acid esterified to Fibric acidcompounds, especially the naturally occurring amino acids, arepharmaceutically elegant free flowing powders, and are rapidly absorbedinto the body. If cleaved in the body, they release non-toxic aminoacids upon cleavage in the body. However, the amino acid derivatives offibric acid have the same utility as the fibric acid compounds fromwhich they are prepared and which are not bound to an amino acid. Theyrequire none of the emulsifiers, additives and other excipients.

Furthermore, the present inventor has found that the amino acidderivatives of Fibric acid derivatives are highly effectiveanti-hyperlipidemics and exhibit such effect intact. The amino acidderivatives are effective anti-hyperlipidemics and are useful in thetreatment of a number of high cholesterol related illnesses and exhibitsuch potential with or without releasing the active parent drug.

While the amino acid derivatives of fibric acid of the present inventionare not expected to possess any acidic activity due to blockage of thecarboxylic acid group responsible for such, the present inventor hasfound that the amino derivatives of fibric acid are effectiveanti-hyperlipidemics with or without releasing Fibric acid derivatives.However, without wishing to be bound, it is believed that Fibric acidamino acid derivatives described herein may or may not release in vivothe active drug with all its pharmacological and cholesterol loweringproperties.

The present invention clearly provides a number of advantages overFibric acid compounds, for example, all of the side chains cleaved invivo, if at all, from these derivatives are naturally occurringessential amino acids and hence are non-toxic. This results in hightherapeutic index. Secondly the amino acid derivatives may be readilycleaved in the body to release Fibric acid or its active component. Onthe other hand, the fibric acid amino acid derivatives of the presentinvention exhibit the same therapeutic properties as the fibric acidcompounds from which they are found thereon. Furthermore, due to theirhigh water solubility, the amino acid derivatives can be easilyadministered by either forming an in-situ solution just before IVadministration using lyophilized sterile powder or providing the drug insolution in prefilled syringe or bottles for infusion. The amino acidesters are more stable than Fibric acid compounds since the COOH groupin Fibric acid is blocked to reaction with bases. Thus the Fibric acidamino acid derivatives described here are more effective then Fibricacid derivatives itself without the toxicity and other pharmaceuticalproblems associated with current marketed formulations.

The amino acid derivatives of this invention are anti-hyperlipidemicdrugs useful in the treatment of hyperlipidemia in mammals where thesymptoms are elevated triglycerides, low HDL (High density lipoproteinsor “good” cholesterol, and elevated cholesterol. The Fibric Acid aminoacid derivatives are also useful in reducing LDL (Low densitylipoproteins, or “bad” cholesterol).

Typical examples of synthesis of L-threonine, L-hydroxyproline andL-serine esters of Fibric acid derivatives are shown in the syntheticprocesses outlined below. These procedures are applicable to all othercompounds of the Fibric acid derivatives class as well.

Synthesis of Fibric Acid Derivatives Derivatives

The procedure for the synthesis of the L-serine, L-threonine, andL-hydroxyproline esters of fenofibric acid is outlined in SyntheticSequence section and is exemplary. The complete procedure and analyticaldata is given in the Experimental Section. In general, fenofibric acid(100 g batches) was prepared from 4-chloro-4′-hydroxybezophenone inaccordane with the procedures in the literature. Fenofibric acid wascoupled with the t-butyl esters of N-Boc protected amino acid (L-serine,L-threonine, and L-hydroxyproline) using EDC as the coupling agents anda catalytic amount of DMAP. The protecting groups were removed at lowtemperature (5° C., 3-6 days) with a mixture of hydrochloric acid inacetic acid (1M) with dichloromethane. The amino acid ester salts offenofibric acid were purified by crystallization from ethyl acetate, anddried under high vacuum.

Synthetic Sequence:

Synthesis of the L-Serine, L-Threonine, and L-Hydroxyproline Esters ofFenofibric Acid a) Boc-Ser-OtBu, EDC, DMAP, CH₂Cl₂; b) Boc-Thr-OtBu,EDC, DMAP, CH₂Cl₂; c) Boc-Hyp-OtBu, EDC, DMAP, CH₂Cl₂; d) HCl, AcOH,CH₂Cl₂ Experimental Section

The synthesis of SPIB00201, SPIB00202 and SPIB00203 was conducted in oneor two batches. Reagents mentioned in the experimental section werepurchased at the highest obtainable purity from Lancaster,Sigma-Aldrich, Acros, or Bachem, except for solvents, which werepurchased from either Fisher Scientific or Mallinkrodt.

1) Synthesis of Fenofibric Acid

A mixture of 4-chloro-4′-hydroxybezophenone (116 g, 0.500 mole) andsodium hydroxide (120 g, 3.00 mole) in acetone (1 L) was heated toreflux for 2 hours. The heating was stopped and the heating source wasremoved. A mixture of chloroform (179 g, 1.50 mole) in acetone (300 mL)was added drop-wise. The reaction mixture was stirred overnight withoutheating. The mixture was heated to reflux for 8 hours and then allowedto cool to room temperature. The precipitate was removed by filtrationand washed with acetone (100 mL). The filtrate was concentrated underreduced pressure to give a brown oil. Water (200 mL) was added to thebrown oil and was acidified (to pH=1) with 1N hydrochloric acid. Theprecipitate, which formed was filtered and dried under high vacuum. Theremaining yellow solid (268 g) was recrystallized from toluene in 4batches (400 mL toluene each). After filtration and drying under highvacuum, the fenofibric acid (116 g, 73% yield) was obtained as a lightyellow solid.

¹H NMR (300 MHz, DMSO-d₆): δ=13.22 (1H, s, br), 7.72 (4H, d, J=8.4 Hz),7.61 (2H, d, J=7.8 Hz), 6.93 (2H, d, J=7.8 Hz), 1.60 (6H, s).

¹³C NMR (75 MHz, DMSO-d₆): δ=192.96, 174.18, 159.35, 136.84, 136.12,131.67, 131.02, 129.12, 128.43, 116.91, 78.87, 25.13.

2) SPIB00201 L-serine-fenofibric acid ester

To a mixture of fenofibric acid (11.6 g, 36.3 mmol),N-carbobenzyloxy-L-serine t-butyl ester (Boc-Ser-OtBu, 8.62 g, 33.0mmol), EDC (7.59 g, 39.6 mmol), and DMAP (484 mg, 3.96 mmol) cooled inan ice-water bath was added anhydrous dichloromethane (150 mL) dropwise.After the addition was complete, the ice bath was removed and thereaction mixture was stirred under an argon atmosphere at roomtemperature for 20 hours. After 20 hours, the additional dichloromethane(200 mL) was added, and the solution was washed with water (2×200 mL)and brine (200 mL). After drying over sodium sulfate and filtration, thesolution was concentrated under reduced pressure. The remaining yellowoil (21.2 g) was purified by column chromatography on silica gel (400 g,0.035-0.070 mm, 6 nm pore diameter), eluting with heptane/ethyl acetate(3:1). After concentration of the product-containing fractions underreduced pressure and drying under high vacuum until the weight wasconstant, the experiment produced the protected L-serine-fenofibric acidester SPIB0020101 (16.2 g, 87% yield) was obtained as a light yellowoil.

¹H NMR (300 MHz, CDCl₃): δ=7.75 (2H, d, J=9.0 Hz), 7.72 (2H, d, J=9.0Hz), 7.45 (2H, d, J=8.7 Hz), 6.86 (2H, d, J=8.7 Hz), 5.04 (1H, d, J=6.9Hz), 4.55-4.42 (3H, m), 1.66 (3H, s), 1.65 (3H, s), 1.43 (9H, s), 1.39(9H, s).

¹³C NMR (75 MHz, CDCl₃): δ=193.92, 172.99, 168.07, 159.24, 154.87,138.24, 136.19, 131.94, 131.06, 130.40, 128.41, 117.26, 82.88, 80.13,79.24, 65.44, 53.44, 28.27, 27.92, 25.70, 25.30.

To a stirred solution of the protected L-serine-fenofibric acid esterSPIB0020101 (16.2 g, 28.8 mmol) in anhydrous dichloromethane (100 mL)cooled to 5° C., under an argon atmosphere was added a solution ofhydrogen chloride in acetic acid (400 mL, 1M, 400 mmol) drop-wise. Thereaction mixture was stirred for 3 days at 5° C. After three days, themixture was concentrated under reduced pressure and dried under highvacuum to remove acetic acid. To the remaining light yellow oil (24.7 g)was added ethyl acetate (100 mL). The solution was concentrated anddried a second time. To the remaining light yellow oil (17.0 g) wasadded ethyl acetate (65 mL). The mixture was heated to reflux for 5minutes and cooled to room temperature. The precipitate was removed byfiltration and dried under high vacuum overnight at room temperature,then at 43° C. for one hour. The experiment produced theL-serine-fenofibric acid ester, hydrochloride SPIB00201 (7.66 g, 60%yield) was obtained as a white solid.

¹H NMR (300 MHz, DMSO-d₆): δ=14.12 (1H, s, br), 8.77 (3H, s, br), 7.72(4H, m), 7.62 (2H, d, J=8.4 Hz), 6.92 (2H, d, J=9.0 Hz), 4.62 (1H, dd,J=12.0, 4.2 Hz), 4.50 (1H, dd, J=12.0, 2.4 Hz), 4.41 (1H, m), 1.64 (3H,s), 1.63 (3H, s).

¹³C NMR (75 MHz, DMSO-d₆): δ=193.06, 171.70, 168.06, 158.72, 136.93,136.06, 131.73, 131.09, 129.62, 128.49, 117.64, 79.02, 62.99, 51.11,25.04, 24.94.

HPLC Analysis:

100% purity; r.t.=4.361 min.; 55% TFA (0.1%), 45% ACN; 1 mL/min; 32.3 C,Luna C18, serial #167917-13; 20 ul inj., NB275-49.

CHN Analysis:

calc.: C, 54.31; H, 4.79; N, 3.17. found: C, 54.37; H, 4.78; N, 3.12.

Melting point: 151° C. (dec.)

3) SPIB00202 L-threonine-fenofibric acid ester

To a mixture of fenofibric acid (25.5 g, 79.9 mmol),N-carbobenzyloxy-L-threonine t-butyl ester (Boc-Thr-OtBu, 20.0 g, 72.6mmol, prepared by the literature method), EDC (16.7 g, 87.1 mmol), andDMAP (1.06 g, 8.71 mmol) cooled in an ice-water bath was added anhydrousdichloromethane (200 mL), dropwise. After the addition was complete, theice bath was removed and the reaction mixture was stirred under an argonatmosphere at room temperature for 20 hours. After 20 hours, additionalEDC (1.39 g, 7.26 mmol) was added, and the reaction mixture was allowedto stir over the weekend at room temperature under an argon atmosphere.After 4 days, additional dichloromethane (300 mL) was added, and thesolution was washed with water (300 mL) and brine (300 mL). After dryingover sodium sulfate and filtration, the solution was concentrated underreduced pressure. The remaining yellow oil (53.5 g) was purified bycolumn chromatography on silica gel (500 g, 0.035-0.070 mm, 6 nm porediameter), eluting with heptane/ethyl acetate (3:1). After concentrationof the product-containing fractions under reduced pressure and dryingunder high vacuum until the weight was constant, the protectedL-threonine-fenofibric acid ester SPIB0020201 (34.1 g; 82% yield) wasobtained as a white foam.

¹H NMR (300 MHz, CDCl₃): δ=7.74 (2H, d, J=8.4 Hz), 7.72 (2H, d, J=8.4Hz), 7.45 (2H, d, J=8.4 Hz), 6.87 (2H, d, J=8.4 Hz), 5.47 (1H, m), 4.98(1H, d, J=9.9 Hz), 4.31 (1H, d, J=9.9 Hz), 1.65 (3H, s), 1.64 (3H, s),1.45 (9H, s), 1.42 (9H, s), 1.22 (3H, d, J=6.3 Hz).

¹³C NMR (75 MHz, CDCl₃): δ=193.94, 172.14, 168.70, 159.26, 155.62,138.28, 136.18, 131.90, 131.08, 130.37, 128.43, 117.40, 82.70, 80.17,79.38, 72.02, 57.46, 28.30, 27.99, 26.44, 24.79, 16.90.

To a stirred solution of the protected L-threonine-fenofibric acid esterSPIB0020201 (34.1 g, 59.2 mmol) in anhydrous dichloromethane (100 mL)cooled to 5° C., under an argon atmosphere was added a solution ofhydrogen chloride in acetic acid (600 mL, 1M, 600 mmol) drop-wise. Thereaction mixture was kept for 6 days at 5° C. The mixture wasconcentrated under reduced pressure and dried under high vacuum toremove acetic acid. To the remaining white solid (45.8 g) was addedethyl acetate (500 mL). The mixture was heated to reflux for 10 minutesand cooled to room temperature. The precipitate was removed byfiltration and dried under high vacuum overnight at room temperature,yielding the L-threonine-fenofibric acid ester, hydrochloride SPIB00202(26.3 g, 97% yield) as a white solid.

¹H NMR (300 MHz, DMSO-d₆): δ=14.10 (1H, s, br), 8.84 (3H, s, br), 7.73(4H, m), 7.63 (2H, d, J=8.1 Hz), 6.89 (2H, d, J=8.7 Hz), 5.44 (1H, m),4.31 (1H, s), 1.64 (3H, s), 1.62 (3H, s), 1.38 (3H, d, J=6.3 Hz).

¹³C NMR (75 MHz, DMSO-d₆): δ=193.04, 171.00, 168.13, 158.76, 136.90,136.08, 131.70, 131.06, 129.49, 128.48, 117.41, 78.99, 69.40, 55.21,25.59, 24.22, 16.06.

HPLC Analysis:

98.59% purity; r.t.=4.687 min.; 55% TFA (0.1%), 45% ACN; 1 mL/min; 32.3C, Luna C18, serial #167917-13; 20 ul inj., NB275-49, DAD1 B, Sig=210.4,Ref=550, 100.

CHN Analysis:

calc.: C, 55.27; H, 5.08; N, 3.07. found: C, 54.98; H, 5.13; N, 3.03.

Melting point: 160.5° C. (dec.)

4) SPIB00203 L-hydroxyproline-fenofibric acid ester

To a mixture of fenofibric acid (24.9 g, 78.1 mmol),N-carbobenzyloxy-L-hydroxyproline t-butyl ester (Boc-Hyp-OtBu, 20.4 g,71.0 mmole, prepared in accordance with the procedure in theliterature), EDC (16.3 g, 85.2 mmol), and DMAP (1.04 g, 8.52 mmol)cooled in an ice-water bath was added anhydrous dichloromethane (200 mL)dropwise. After the addition was complete, the ice bath was removed andthe reaction mixture was stirred under an argon atmosphere at roomtemperature for 20 hours. After 20 hours, additional EDC (1.63 g, 8.52mmol) was added and the experiment was allowed to stir over the weekendat room temperature under an argon atmosphere. After 4 days, theresulting solution was washed with water (200 mL) and brine (200 mL).After drying over sodium sulfate followed by filtration, the solutionwas concentrated under reduced pressure. The remaining yellow oil (49.4g) was purified by column chromatography on silica gel (500 g,0.035-0.070 mm, 6 nm pore diameter), eluting with heptane/ethyl acetate(2:1). After concentration of the product containing fractions underreduced pressure and drying under high vacuum until the weight wasconstant, the protected L-hydroxyproline-fenofibric acid esterSPIB0020301 (26.4 g, 63% yield) was obtained as a colorless oil.

¹H NMR (300 MHz, CDCl₃): δ=7.76 (2H, d, J=8.1 Hz), 7.73 (2H, d, J=8.1Hz), 7.46 (2H, d, J=8.1 Hz), 6.84 (2H, d, J=8.1 Hz), 5.32 (1H, m), 4.13(0.38H, t, J=7.8 Hz), 4.00 (0.62H, t, J=7.8 Hz), 3.67 (1.62H, m), 3.46(0.38H, d, J=12.6 Hz), 2.29 (1H, m), 2.15 (1H, m), 1.68 (3H, s), 1.66(3H, s), 1.44-1.38 (18H, m).

¹³C NMR (75 MHz, CDCl₃): δ=193.88, 172.98, 171.14, 159.25, 153.48,138.23, 136.16, 131.99, 131.08, 130.36, 128.44, 117.03, 116.91, 81.48,80.32, 80.20, 79.19, 74.03, 73.26, 58.23, 51.88, 51.58, 36.33, 35.31,31.92, 28.29, 28.00, 25.89, 24.95.

To a stirred solution of the protected L-hydroxyproline-fenofibric acidester SPIB0020301 (26.0 g, 44.2 mmol) in anhydrous dichloromethane (100mL) cooled to 5° C., under an argon atmosphere was added a solution ofhydrogen chloride in acetic acid (450 mL, 1M, 450 mmol) drop-wise. Thereaction mixture stirred for 4 days at 5° C. After four days the mixturewas concentrated under reduced pressure and dried under high vacuum toremove acetic acid. To the remaining yellow oil (31.5 g) was added ethylacetate (200 mL). The mixture was sonicated and then concentrated underreduced pressure and dried under high vacuum. To the remaining whitesolid (23.2 g) was added ethyl acetate (300 mL). The ethyl acetatemixture was heated to reflux for 10 minutes, and cooled to roomtemperature. The precipitate was removed by filtration and dried underhigh vacuum overnight at room temperature. L-hydroxyproline-fenofibricacid ester, hydrochloride SPIB00203 (15.8 g, 76% yield) was obtained asa white solid.

¹H NMR (300 MHz, DMSO-d₆): δ=14.07 (1H, s, br), 10.75 (1H, s, br), 9.40(1H, s, br), 7.71 (4H, d, J=8.1 Hz), 7.60 (2H, d, J=8.1 Hz), 6.96 (2H,d, J=8.1 Hz), 5.42 (1H, m), 4.24 (1H, t, J=9.0 Hz), 3.61 (1H, dd,J=13.2, 4.2 Hz), 3.28 (1H, d, J=13.2 Hz), 2.35 (2H, m), 1.66 (3H, s),1.64 (3H, s).

¹³C NMR (75 MHz, DMSO-d₆): δ=193.00, 171.52, 169.14, 158.81, 136.87,136.09, 131.81, 131.05, 129.48, 128.46, 117.28, 78.99, 73.79, 57.54,50.23, 34.13, 25.69, 24.49.

HPLC Analysis:

100% purity; r.t.=8.369 min.; 60% DIUF water (0.1% TFA)/40%acetonitrile; 1 mL/min; 36.4 C; Luna C18, 5 u column (serial #191070-3),4.6×250 mm; 20 ul injection; DAD1 A, Sig=210.4, Ref=550, 100.

HPLC-MS (ESI): calculated: M⁺⁼431. found M+H=432.3

Melting point: 187.5° C. (dec.)

Solubility of the above esters were determined in water at roomtemperature by dissolving an excess of each of the drug and permittingeach to settle for a few hours. The resulting solutions were centrifugedat 1500 rpm for 3 min and the supernatant liquid was analyzed. Byproviding the memory of Sikihn, Esq. esters posses solubility in waterin excess of 50 mg/mL.

EXPERIMENTAL

Rats were checked for time zero triglyceride level in blood. Then therats were set on high sugar diet, such as 30% surcorse in water for 1week. Then at the end of 1 week, rats were tested for triglycerides, andwere put on normal diet. From day 7-14 the rats were administered eithertest or control drug. Triglycerides were again tested on the 14^(th) dayin rat blood.

In the Fenofibrate (control) vs L-Serine Ester of Fenofibric acid (testdrug), 3 rats each for each of the drug and control at equivalent dosesof 50, 100 and 200 mg/kg were tested.

The results are shown below in Tables 35 and 36.

TABLE 35 SUMMARY - DOSE RANGE FINDING STUDY - HYPOLIPIDEMIC PROPERTY -FENOFIBRATE AND ITS FORMULATION Dose. Triglycerides (mg/dl) Test ItemMg/kg) Animal No. Day zero Day 7 Day 14 Vehicle 0 1 81 168 121 2 88 171222 3 114 133 162 Reference control 50 4 95 157 101 Fenofibrate 5 92 22876 6 80 150 73 100 7 110 204 62 8 115 195 69 9 96 167 93 200 10 144 9048 11 56 106 51 12 58 125 38 L-Serine Ester of 50 13 88 148 86Fenofibric Acid 14 94 145 86 15 100 127 73 100 16 109 — 46 17 129 100 6918 71 183 47 200 19 74 240 83 20 81 158 61 21 42 77 46 Test Substance:L-Serine Ester of Fenofibric Acid Vehicle: 1% Tween 80 in milli Q -water

TABLE 36 Anti-lipidemic Effects of Fenofibric Acid and its Derivatives %% Absolute Absolute Change Change Change Change From from from day fromday day day Dose 0 7 0 7 Vehicle mg/kg 50.4 −5.6  83.17%  −4.80% Fen-S25 −54 −90 −61.64% −72.82% Ester 50 −31.4 −84.2 −44.10% −67.90% 100−20.8 −72 −33.23% −63.27% Fen-T 25 −18.2 −36.4 −23.21% −37.68% Ester 50−23.8 −77.6 −35.00% −63.71% 100 −63.8 −88.4 −68.45% −75.04% Fen-HP 25−16 −47.8 −32.92% −59.45% Ester 50 −35.8 −70.8 −49.31% −65.80% 100 −3.4−112  −7.52% −72.82% Fenofibrate 25 −10.8 −51 −15.21% −45.86% 50 −13.4−87.6 −22.95% −66.06% 100 −40.8 −71.6 −61.26% −73.51%

While all of the esters were active and showed efficacy, there wereimportant distinguishing factors between various amino acid derivativesand Fenofibrate. For example, dose dependent decrease in triglycerideswere noted with the L-Threonine ester, and also maximum decrease frombaseline level and treatment level were also noted for this compound.Thus Fenofibric Acid-L-Threonine ester had overall superioranti-hyperlipidemic properties.

From the above results, it can be concluded that both the highly watersoluble serine ester, L-threonine ester and hydroxyproline estereffectively performed compared to fenofibrate.

There are a number of screening tests to determine the utility of theamino acid derivatives created according tio the disclosed methods.These include both in vitro and in vivo screening methods.

The in vitro methods include acid/base hydrolysis of the amino acidderivatives, e.g., hydrolysis in pig pancreas hydrolysis in ratintestinal fluid, hydrolysis in human gastric fluid, hydrolysis in humanintestinal fluid, and hydrolysis in human blood plasma. These assays aredescribed in Simmons, D M, Chandran, V R and Portmann, G A, DanazolAmino Acid Derivatives: In Vitro and In Situ BiopharmaaceuticalEvaluation, Drug Development and Industrial Pharmacy, Vol 21, Issue 6,Page 687, 1995, the contents of all of which are incorporated byreference.

The amino acid derivatives of Fibric Acid of the present invention areeffective in treating diseases or conditions in which Fibric acidderivatives normally are used. The amino acid derivatives disclosedherein are transformed within the body to potentially release the activecompound, although the active agent in vivo may be the intact amino acidderivative of fibric acid. In addition, the amino acid derivatives offibric acid also enhance the therapeutic benefits of the Fibric acidcompounds by reducing or eliminating biopharmaceutical andpharmacokinetic barriers associated with each of them. However it shouldbe noted that these amino acid derivatives themselves will havesufficient activity without releasing any active drug in the mammals.

Thus, the amino acid derivatives of the present invention enhance thetherapeutic benefits by removing biopharmaceutical and pharmacokeneticbarriers of existing drugs.

Furthermore, the amino acid derivatives are easily synthesized in highyields using reagents which are readily and commercially available.

In the formula hereinabove, it is to be understood that the AA has thefollowing definition in the following context unless indicated to thecontrary

AA in this definition refers to the amino acid residue without an aminogroup either on the main chain or the side chain.

AA in this definition is an amino acid residue less the hydroxy group onthe side chain.

AA refers to an amino acid group without the carboxy group, either onthe main chain or side group.4) OAA—This is a ester bond between the hydroxy group of the drug andthe carboxy group of the amino acid either on the main chain or sidechain. Thus, as written OAA is

wherein R_(O) is the side chain amino acid as defined hereinabove.

Alternatively, it may refer to an ester bond between the carboxy groupof the drug and the hydroxy group on the side chain of those amino acidswhich have a hydroxy group thereon such as threonine, serine,hydroxyproline, tyrosine and the like. The hydroxy group forms part ofthe ester linkage which is depicted hereinabove with O. Thus, aswritten, the AA refers to an amino acid with a hydroxy group on the sidechain, but as depicted as OAA, the AA is without the hydroxy group sincethe oxygen atom is depicted in the formula.

As used herein, the term “amino acid derivative” or equivalent termthereto refers to amino acid moiety covalently bonded to the drug havinga functional group thereon consisting of hydroxy, amino, carboxy andacylating derivatives of carboxy.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A compound or the pharmaceutically acceptable salt thereof, wherein adrug having a functionality selected from the group consisting ofhydroxy, amino, carboxy or acylating derivative of said carboxy group isbonded to an amino acid wherein the drug is selected from the consistingof 5-HETE, Abacavir, Acarbose, Acebutolol, Acetaminophen, Adefovir,Albuterol, Alfaprostol acid, Amlodipine, Amoxicillin, Amphotericin B,Amprenavir, Arachidonic Acid, Aspirin, Atenolol, Atorvastatin, Atropine,Atovaquone, Baclofen, Benazeprilat, Beraprost, Bexarotene, Bicalutamide,Biperiden, Bisoprolol, Bitolterol, Brinzolamide, Bupivacaine,Buprenorphine, Bupropion, Butorphanol, Candesartan, Capacitabine,Captopril, Carbidopa, Carboprost, Carnitine, Carteolol, Carvedilol,Cefdinir, Cefditoren, Ceftazimide, Cefpodoxime, Cefuroxime,Cerivastatin, Chloramphenicol, Cisapride, Clofibrate, Cloprostenol,Clorazepic Acid, Cycloserine, Cyclosporine, Cytarabine,Dextroamphetamine, Diclofenac, Didanosine, Divalproex, Efavirenz,Enalaprilat, Ephedrine, Eplerenone, Eprosartan, Esmolol, Estramustine,Ethambutol, Ethchlorvynol, Ethotoin, Etidocaine, Etoposide, Ezetimibe,Famciclovir, Fenofibrate, Fenoprofen, Fenprostalene acid, Fibric acidderivatives, Finasteride, Flavoxate, Fluprostenol, Fluoxetine,Flurbiprofen, Fluticasone, Fluvastatin, Fosinoprilat, Frovatriptan,Fulvestrant, Gemprost Acid, Goserelin, Hydroxychloroquine, Hydroxyzine,Hyoscyamine, Ibuprofen, Ibutilide, Indapamide, Indinavir, Ipratropium,Irinotecan, Isosorbide, Isradipine, Ketoprofen, Ketorolac, Labetalol,Lamivudine, Lansoprazole, Latanoprost Acid, Leukotrienes (LTA₄, LTB₄,LTC₄, LTD₄ and LTE₄) Leuprolide, Levobunolol, Levodopa, Levorphanol,Limaprost, γ-Linolenic Acid, Liothyronine, Lisinopril, Lopinavir,Lorazepam, Lovastatin, Medroxyprogesterone, Mefloquine, Megestrol,Mephobarbital, Mepivacaine, Metaproterenol, Metformin, Methamphetamine,Methohexital, Methotrexate, Methylprednisolone, Metolazone, Metoprolol,Mexiletine, Miglitol, Moexiprilat, Mometasone, Montelukast,Mycophenolate Acid, Nadolol, Nalbuphine, Naproxen, Naratriptan,Nateglinide, Nelfinavir, Niacin, Nicotinic Acid, Nicotinamide,Nicardipine, Nimidipine, Nisoldipine, Norgestimate, Octreotide,Ofloxacin, Olmesartan, Omeprazole, Ozagrel, Paclitaxel, PantothenicAcid, Paroxetine, Penbutolol, Penciclovir, Pentazocine, Pentobarbital,Perindoprilat, Phenylephrine, Phenylpropanolamine, Pindolol,Pioglitazone, Pirbuterol, Pramipexole, Pravastatin, Propafenone,Propofol, Propoxyphene, Propranolol, Prostacyclin, Prostaglandins (E₁,E₂ and F_(2α)), Prostanoic Acid, Pseudoephedrine, Quinacrine,Quinaprilat, Quinethazone, Quinidine, Quinine, Ramiprilat, Reboxetine,Repaglinide, Ribavirin, Ritonavir, Ropivacaine, Rosaprostol,Rosuvastatin, Salmeterol, Salsalate, Sertraline, Simavastatin, Sotalol,Sulfa Drugs, Sulfasalazine, Sumitriptan, Tacrolimus, Tazorotene,Telmesartan, Tenofovir, Terbutaline, Tiagabine, Timolol, Tirofiban,Tocainide, Tramadol, Trandolaprilat, Tranylcypromine, Treprostinil,Triamcinolone, Trimoprostil, Troglitazone, Unoprostone, Valproic Acid,Valsartan, Venlafaxine, Vidarabine, Warfarin, Zalcitabine, Zidovudine,Zileuton and Zolmitriptan or a pharmaceutically acceptable salt of anyof said drug and wherein the amino acid is L-Hyp, L-Ser, L-Tyr, L-Lys,L-Leu, L-Ile, Gly, L-Asp, L-Glu, L-Met, L-Ala, L-Val, L-Pro, L-His,L-Nor, L-Arg, L-Phe, L-Trp, L-Car, L-Ort, GABA, L-Cys, or Thr.
 2. Thecompound according to claim 1 wherein the drug is Acarbose, Glimepiride,Metformin, Miglitol, Nateglinide, Pioglitazone, Repaglinide, orTroglitazone.
 3. The compound according to claim 1 wherein the drug isAdefovir, Amoxicillin, AmphotericinB, Amprenavir, Abacavir, Ethambutol,Atovaquone, Cefdinir, Cefditoren, Ceftazimide, Cefpodoxime, Cefuroxime,Chloramphenicol, Quinine, Tenofovir, Vidarabine, Zalcitabine,Zidovudine, Mefloquine, Quinidine, Didanosine, Efavirenz, Famciclovir,Sulfa Drugs, Ribavirin, Ritonavir, Indinavir, Lamivudine, Lopinavir,Nelfinavir, Cycloserine, Ofloxacin or Penciclovir.
 4. The compoundaccording to claim 1 wherein the drug is Amlodipine, Acebutolol,Atenolol, Benazeprilat, Beraprost, Candesartan, Captopril, Bisoprolol,Carvedilol, Enalaprilat, Ephedrine, Eplerenone, Eprosartan, Esmolol,Fibric acid derivatives, Fosinoprilat, Isradipine, Metolazone,Labetalol, Metoprolol, Moexiprilat, Isosorbide, Mexiletine, Nadolol,Fenofibrate, Indapamide, Lisinopril, Nicardipine, Nisoldipine,Olmesartan, Penbutolol, Perindoprilat, Pindolol, Sotalol, Propafenone,Propranolol, Prostacyclin, Prostaglandins (EI, E2 and F_(2α)),Prostanoic Acid, Quinaprilat, Quinethazone, Ramiprilat, Telmisartan,Phenylephrine, Tirofiban, Tocainide, Trandolaprilat, Valsartan, orIbutilide.
 5. The compound according to claim 1 wherein the drug isRosuvastatin, Simavastatin, Fluvastatin, Cerivastatin, Pravastatin,Ezetimibe, Nicotinic Acid, Niacin, Clofibrate, Lovastatin orAtorvastatin.
 6. The compound according to claim 1 wherein the drug isAspirin, Butorphanol, Acetaminophen, Diclofenac, Fenoprofen,Flurbiprofen, Frovatriptan, Ibuprofen, Ketoprofen, Ketorolac,Levorphanol, Methylprednisolone, Nalbuphine, Naproxen, Naratriptan,Nicotinamide, Pentazocine, Propoxyphene, Salsalate, Sumitriptan,Tramadol or Zolmitriptan.
 7. The compound according to claim 1 whereinthe drug is Finasteride, 5-HETE, Bicalutamide, Cytarabine, Estramustine,Capecitabine, Estramustine, Goserelin, Fulvestrant, Etoposide,Leuprolide, Alfaprostol acid, Megestrol, Methotrexate or Paclitaxel. 8.The compound according to claim 1 wherein the drug is Biperiden,Carbidopa, Levodopa, Pramipexole, or Atropine.
 9. The compound accordingto claim 1 wherein the drug is Baclofen, Limaprost, Fluprostenol,Brinzolamide, Carteolol, Latanoprost Acid, Gemprost Acid, Levobunolol,Timolol or Unoprostone.
 10. The compound according to claim 1 whereinthe drug is Clorazepic Acid, Buprenorphine, Bupropion, Fluoxetine,Lorazepam, Pantothenic Acid, Paroxetine, Sertraline, Venlafaxine,Phenylpropanolamine, Valproic Acid, Reboxetine or Tranylcypromine. 11.The compound according to claim 1 wherein the drug is Ethotoin,Mephobarbital, Pentobarbital or Tiagabine.
 12. The compound according toclaim 1 wherein the drug is Bitolterol, Albuterol, Hydroxyzine,Fexofenadine, Fluticasone, Ipratropium, Ozagrel, Leukotrienes (LTA₄,LTB₄, LTC₄, LTD₄ and LTE₄), Metaproterenol, Montelukast, Mometasone,Pirbuterol, Pseudoephedrine, Salmeterol, Terbutaline, Treprostinil orZileuton.
 13. The compound according to claim 1 wherein the drug isFlavoxate, Hyoscyamine, Lansoprazol, Cisapride, Omeprazole, Octreotide,Rosaprostol, Trimoprostil or Irinitecan.
 14. The compound according toclaim 1 wherein the drug is Hydroxychloroquine, Sulfasalazine orQuinacrine.
 15. The compound according to claim 1 wherein the drug isNorgestimate or Medroxyprogesterone.
 16. The compound according to claim1 wherein the drug is Etidocaine, Ethchlorvynol, Mepivacaine,Methohexital, Propofol, Ropivacaine, or Bupivacaine.
 17. The compoundaccording to claim 1 wherein the drug is Methamphetamine,Dextroamphetamine or Pemoline.
 18. The compound according to claim 1wherein the drug is Mycophenolate Acid or Cyclosporine.
 19. The compoundaccording to claim 1 wherein the drug is Bexarotene, Tacrolimus,Tazorotene or Triamcinolone.
 20. The compound according to claim 1wherein the drug is Nimodipine, Carboprost, Carnitine or Warfarin. 21.The compound according to claim 1 wherein the drug is Arachidonic Acid,Linolenic Acid or Liothyronine.
 22. The compound according to claim 1wherein the drug is Fenprostalene acid, or Cloprostenol.
 23. Thecompound according to claim 1 wherein the drug is Atorvastatin, Aspirin,Amlodipine Albuterol, Valsartan, Albuterol, Ipratropium, Olmesartan,Abacavir, Lamivudin, Adefovir, Buprenorphine, Benazeprilat, Bupriopion,Clopidogrel, Candesartan, Cefuroxime, Cerivastatin, Clofibrate,Ciprofloxacine, dexamethorphan, Carvedilol, Cyclosporine, Capacitabine,Divalproex, Dextroamphitamine, Diclofenac, Enalaprilat, Eprosartan,Efafirenz, Tenofovir, Ezitimible, Simvastatin, Tenovofir, Famciclovir,Fenofibrate, Fluvastaatin, Fosinoprilat, Fulvestrant, FluticasoneSalmenterol, Fenofibrate, Fluticasone, Fenofibrate, Finasteride,Flucasone+Salmet, Fluticason, Goserelin, Hydroxychloroquine, Ibuprofen,Irinotecan, Ketoprofen, Ketorolac, Labetalol, Lansoprazole, LatanoprostAcid, Lorazepam, Lovastatin, Lopinavir, Ritonavir, Lanzoprazol,MedroxyProgesterone, Mometazone, Metformin, Montelukast,Methylphenidate, Metalazone, Methylphenidate, Mycophenolate, Naproxen,Naratriptan, Nelfinavir, Nicotinamide, Niacin, Olmesartan, Pravastatin,Propofol, Pioglitazone, Repaglinide, Rosuvastatin, Ritonavir,Salmeterol, Simavastatin, Sumitriptan, Sulfa Drugs, Trandolaprilat,Tacrolimus, Telmesartan, Tenofovir, Triamcinolone, Valsartan,Venlafaxine, Valproic Acid, Warfarin, Zolmitriptan, or Zidovudin. 24.The compound according to claim 1 or 23 wherein the amino acid is GABA.25. The compound according to claim 1 or 23 wherein the amino acid isL-Ort.
 26. The compound according to claim 1 or 23 wherein the aminoacid is L-Car.
 27. The compound according to claim 1 or 23 wherein theamino acid is L-Trp.
 28. The compound according to claim 1 or 23 whereinthe amino acid is L-Phe.
 29. The compound according to claim 1 or 23wherein the amino acid is L-Arg.
 30. The compound according to claim 1or 23 wherein the amino acid is L-His.
 31. The compound according toclaim 1 or 23 wherein the amino acid is L-Pro.
 32. The compoundaccording to claim 1 or 23 wherein the amino acid is L-Pro.
 33. Thecompound according to claim 1 or 23 wherein the amino acid is L-Val. 34.The compound according to claim 1 or 23 wherein the amino acid is L-Ala.35. The compound according to claim 1 or 23 wherein the amino acid isL-Met.
 36. The compound according to claim 1 or 23 wherein the aminoacid is L-Glu.
 37. The compound according to claim 1 or 23 wherein theamino acid is L-Asp.
 38. The compound according to claim 1 or 23 whereinthe amino acid is Gly.
 39. The compound according to claim 1 or 23wherein the amino acid is L-Ile.
 40. The compound according to claim 1or 23 wherein the amino acid is L-Leu.
 41. The compound according toclaim 1 or 23 wherein the amino acid is L-Lys.
 42. The compoundaccording to claim 1 or 23 wherein the amino acid is L-Tyr.
 43. Thecompound according to claim 1 or 23 wherein the amino acid is L-Ser. 44.The compound according to claim 1 or 23 wherein the amino acid is L-Hyp.45. The compound according to claim 1 or 23 wherein the amino acid isL-Thr.
 46. A compound of the formula:

or pharmaceutically acceptable salts thereof; wherein CYCLO representsthe residues at positions 2-11 of a cyclosporin molecule x-y is CH═CH orCH₂CH₂ and R² is OAA or OGly-AA, AA is an amino acid residue without theOH groups of the carboxy group and where Gly is a glycyl residue. 47.The compound according to claim 46 wherein AA is naturally occurring αL-amino acid.
 48. The compound according to claim 46 wherein AA is Lys,Leu, Ile, Gly, Asp, Glu, Met, Ala, Val, Pro, His, Tyr, Ser, Nor, Thr,Arg, Phe, Trp, Hyp, Hsr, Car, Ort, Cys, Sar or Dcy.
 49. The compoundaccording to claim 46 wherein AA is proline, glycine, lysine orornithine.
 50. The compound according to claim 46 wherein AA is Lys orornithine.
 51. The compound according to claim 46 wherein the compoundhas the formula


52. A pharmaceutical composition comprising a therapeutically effectiveamount of a compound according to claim 46 and a pharmaceuticallyacceptable carrier therefor.
 53. A method of treating a patient in needof cyclosporin therapy, which method comprising administering to saidpatient a therapeutically effective amount of the compound according toclaim
 46. 54. A pharmaceutical composition comprising a therapeuticallyeffective amount of a compound according to claim 1 or 23 and apharmaceutically acceptable carrier therefor.